Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-MERTK ANTIBODIES AND THEIR METHODS OF USE
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S. Provisional
Application Serial Nos.
62/836,580, filed April 19, 2019; and 62/890,858, filed August 23, 2019; each
of which is hereby
incorporated by reference in its entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file is
incorporated herein by
reference in its entirety: a computer readable form (CRF) of the Sequence
Listing (file name:
1463920479405EQLI5T.TXT, date recorded: March 30, 2020, size: 136 KB).
FIELD
[0003] The present disclosure relates to anti-MerTK antibodies and methods
of use thereof.
BACKGROUND
[0004] Currently most cancer immuno-oncology (10) therapies focus on
modulating the activity
of T cells, the adaptive arm of immune system, by blocking inhibitory pathways
that serve as
immunological checkpoints. However, long-lasting responses triggered by these
therapies are limited
to subpopulations of cancer patients. The relatively low response rate is
caused by various
immunosuppressive mechanisms in the tumor microenvironment. The innate immune
system is an
integral part of an effective immune response. Innate immune cells play a
crucial part in initiating and
subsequent direction of the adaptive immune response. Targeting the innate
immune system may
complement the adaptive immuno-oncology therapies (Mullard, A., Nat. Rev. Drug
Discov., 17: 3-5
(2018)).
[0005] Macrophages of the innate immune system are abundant in various
types of solid tumors
and may contribute to the relatively low response rate to T-cell based
therapy. They are versatile cells
capable of carrying out various functions, including phagocytosis. Macrophages
are professional
phagocytes highly specialized in removal of dying or dead cells, and cellular
debris. It is estimated
that billions of cells die every day in the human body. But it is rare to find
apoptotic cells in tissues
under normal physiological conditions thanks to the rapid and efficient
clearance by phagocytes. In
homeostasis, apoptotic cells are removed at the early stage of cell death
before loss of plasma
membrane integrity. Therefore, in general apoptosis is immunologically silent.
In solid tumors,
uncontrolled tumor growth is often accompanied by increased cell death due to
hypoxia and metabolic
stress. To evade immune surveillance, tumors take advantage of the non-
immunogenic nature of
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apoptosis. Tumor associated macrophages (TAMs) actively remove the dying tumor
cells to avoid
alerting the immune system.
[0006] MerTK has been shown to play a role in clearance of apoptotic cells.
Therefore,
reduction of MerTK-mediated clearance of apoptotic cells using MerTK
inhibitors is an attractive
therapeutic approach in treating cancer. Existing anti-MerTK antibodies have
been described but may
be unsuitable for therapeutic development. For example, White et al. ("MERTK-
Specific Antibodies
That Have Therapeutic Antitumor Activity in Mice Disrupt the Integrity of the
Retinal Pigmented
Epithelium in Cynomolgus Monkeys," presented at the American Association for
Cancer Research
Annual Meeting; March 31, 2019; Atlanta, GA) describe two anti-MerTK
antibodies: one that binds
to human MerTK with higher affinity (8.7x10-"M; SRF1), and one that binds to
human MerTK with
lower affinity (4.4x109) but cross-reacts with murine MerTK (SRF2). These
antibodies were shown
to inhibit various MerTK functions and inhibit tumor growth in combination
with anti-PD-Li
antibody in a mouse model. However, both antibodies were found to promote
retinal toxicity in
cynomolgus monkey. As such, neither antibody would be acceptable as a
therapeutic candidate.
These findings underscore the importance of examining multiple factors, not
simply antibody affinity,
in developing an efficacious therapeutic candidate with an acceptable safety
profile.
[0007] Thus, there remains a need for an optimal therapy for treating,
stabilizing, preventing,
and/or delaying development of various cancers. In particular, anti-MerTK
antibodies having optimal
binding characteristics (e.g., on and off rates) as well as desired biological
effects are needed.
[0008] All references cited herein, including patent applications, patent
publications, and
UniProtKB/Swiss-Prot Accession numbers are herein incorporated by reference in
their entirety, as if
each individual reference were specifically and individually indicated to be
incorporated by reference.
SUMMARY
[0009] Described herein are anti-MerTK antibodies and methods of use
thereof that meet the
need for optimized therapy for treating, stabilizing, preventing, and/or
delaying development of
various cancers.
[0010] In one aspect, the present disclosure provides an isolated antibody
that binds to MerTK
where the antibody reduces MerTK-mediated clearance of apoptotic cells. In
some embodiments, the
antibody reduces MerTK mediated clearance of apoptotic cells by phagocytes. In
some embodiments,
the phagocytes are macrophages. In an exemplary embodiment, the macrophages
are tumor-
associated macrophages. In some embodiments, the clearance of apoptotic cells
is reduced as
measured in an apoptotic cell clearance assay at room temperature.
[0011] In some embodiments, anti-MerTK antibodies of the present disclosure
reduce ligand-
mediated MerTK signaling. In some embodiments, the antibodies induce a pro-
inflammatory
response, including but not limited to a type I IFN response.
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[0012] In some embodiments, anti-MerTK antibodies of the present disclosure
are monoclonal
antibodies. In some embodiments, the antibodies are human, humanized, or
chimeric antibodies. In
some embodiments, the antibodies are antibody fragments that bind to MerTK. In
some embodiments,
the antibody binds to a fibronectin-like domain or an immuno globulin-like
domain of MerTK.
[0013] In an exemplary embodiment, an anti-MerTK antibody of the present
disclosure binds to
a fibronectin-like domain of MerTK.
[0014] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to a
fibronectin-like domain of MerTK comprises (a) an HVR-H1 comprising the amino
acid sequence of
SEQ ID NO: 4, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:
5, and (c) an
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In some
embodiments, the
antibody further comprises (a) an HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 1; (b)
an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) an HVR-
L3 comprising
the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibody
comprises (a) a
heavy chain variable domain (VH) comprising a sequence having at least 95%
sequence identity to
the amino acid sequence of SEQ ID NO: 83; (b) a light chain variable domain
(VL) comprising a
sequence having at least 95% sequence identity to the amino acid sequence of
SEQ ID NO: 65; or (c)
a VH as in (a) and a VL as in (b). In some embodiments, the antibody comprises
a VH comprising
the amino acid sequence of SEQ ID NO: 83. In some embodiments, the antibody
comprises a VL
comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the
antibody
comprises a VH comprising the amino acid sequence of SEQ ID NO: 83 and a VL
comprising the
amino acid sequence of SEQ ID NO: 65.
[0015] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to a
fibronectin-like domain of MerTK comprises the antibody comprises (a) an HVR-
H1 comprising the
amino acid sequence of SEQ ID NO: 10, (b) an HVR-H2 comprising the amino acid
sequence of SEQ
ID NO: 11 and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:
12. In some
embodiments, the antibody further comprises (a) an HVR-L1 comprising the amino
acid sequence of
SEQ ID NO: 7; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:
8; and (c) an
HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9. In some
embodiments, the antibody
comprises (a) a heavy chain variable domain (VH) comprising a sequence having
at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 84; (b) a light
chain variable domain
(VL) comprising a sequence having at least 95% sequence identity to the amino
acid sequence of SEQ
ID NO: 66; or (c) a VH as in (a) and a VL as in (b). In some embodiments, the
antibody comprises a
VH comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments,
the antibody
comprises a VL comprising the amino acid sequence of SEQ ID NO: 66. In some
embodiments, the
antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 84
and a VL
comprising the amino acid sequence of SEQ ID NO: 66.
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[0016] In some embodiments, the antibody comprises (a) a heavy chain
variable domain (VH)
comprising a sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID
NO: 85; (b) a light chain variable domain (VL) comprising a sequence having at
least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 67; or (c) a VH as in (a)
and a VL as in (b). In
some embodiments, the antibody comprises a VH comprising the amino acid
sequence of SEQ ID
NO: 85. In some embodiments, the antibody comprises a VL comprising the amino
acid sequence of
SEQ ID NO: 67. In some embodiments, the antibody comprises a VH comprising the
amino acid
sequence of SEQ ID NO: 85 and a VL comprising the amino acid sequence of SEQ
ID NO: 67.
[0017] In some embodiments, the antibody comprises a heavy chain comprising
the amino acid
sequence of SEQ ID NO: 102. In some embodiments, the antibody comprises a
light chain
comprising the amino acid sequence of SEQ ID NO: 110.
[0018] In some embodiments, the antibody comprises (a) a heavy chain
variable domain (VH)
comprising a sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID
NO: 86; (b) a light chain variable domain (VL) comprising a sequence having at
least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 68; or (c) a VH as in (a)
and a VL as in (b). In
some embodiments, the antibody comprises a VH comprising the amino acid
sequence of SEQ ID
NO: 86. In some embodiments, the antibody comprises a VL comprising the amino
acid sequence of
SEQ ID NO: 68. In some embodiments, the antibody comprises a VH comprising the
amino acid
sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ
ID NO: 68.
[0019] In some embodiments, the antibody comprises a heavy chain comprising
the amino acid
sequence of SEQ ID NO: 103. In some embodiments, the antibody comprises a
light chain
comprising the amino acid sequence of SEQ ID NO: 111.
[0020] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to a
fibronectin-like domain of MerTK comprises the antibody comprises (a) an HVR-
H1 comprising the
amino acid sequence of SEQ ID NO: 16, (b) an HVR-H2 comprising the amino acid
sequence of SEQ
ID NO: 17 and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:
18. In some
embodiments, the antibody further comprises (a) an HVR-L1 comprising the amino
acid sequence of
SEQ ID NO: 13; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:
14; and (c) an
HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15. In some
embodiments, the
antibody comprises (a) a heavy chain variable domain (VH) comprising a
sequence having at least
95% sequence identity to the amino acid sequence of SEQ ID NO: 87; (b) a light
chain variable
domain (VL) comprising a sequence having at least 95% sequence identity to the
amino acid sequence
of SEQ ID NO: 69; or (c) a VH as in (a) and a VL as in (b). In some
embodiments, the antibody
comprises a VH comprising the amino acid sequence of SEQ ID NO: 87. In some
embodiments, the
antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 69.
In some
embodiments, the antibody comprises a VH comprising the amino acid sequence of
SEQ ID NO: 87
and a VL comprising the amino acid sequence of SEQ ID NO: 69.
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[0021] In some embodiments, the antibody comprises (a) a heavy chain
variable domain (VH)
comprising a sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID
NO: 88; (b) a light chain variable domain (VL) comprising a sequence having at
least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 70; or (c) a VH as in (a)
and a VL as in (b). In
some embodiments, the antibody comprises a VH comprising the amino acid
sequence of SEQ ID
NO: 88. In some embodiments, the antibody comprises a VL comprising the amino
acid sequence of
SEQ ID NO: 70. In some embodiments, the antibody comprises a VH comprising the
amino acid
sequence of SEQ ID NO: 88 and a VL comprising the amino acid sequence of SEQ
ID NO: 70.
[0022] In some embodiments, the antibody comprises a heavy chain comprising
the amino acid
sequence of SEQ ID NO: 104. In some embodiments, the antibody comprises a
light chain
comprising the amino acid sequence of SEQ ID NO: 112.
[0023] In some embodiments, the antibody comprises (a) a heavy chain
variable domain (VH)
comprising a sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID
NO: 89; (b) a light chain variable domain (VL) comprising a sequence having at
least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 70; or (c) a VH as in (a)
and a VL as in (b). In
some embodiments, the antibody comprises a VH comprising the amino acid
sequence of SEQ ID
NO: 89. In some embodiments, the antibody comprises a VL comprising the amino
acid sequence of
SEQ ID NO: 70. In some embodiments, the antibody comprises a VH comprising the
amino acid
sequence of SEQ ID NO: 89 and a VL comprising the amino acid sequence of SEQ
ID NO: 70.
[0024] In some embodiments, the antibody comprises a heavy chain comprising
the amino acid
sequence of SEQ ID NO: 105. In some embodiments, the antibody comprises a
light chain
comprising the amino acid sequence of SEQ ID NO: 113.
[0025] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to a
fibronectin-like domain of MerTK comprises (a) an HVR-H1 comprising the amino
acid sequence of
SEQ ID NO: 22, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:
23 and (c) an
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In some
embodiments, the antibody
further comprises (a) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 19; (b) an
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) an HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 21. In some embodiments, the antibody
comprises (a) a heavy
chain variable domain (VH) comprising a sequence having at least 95% sequence
identity to the
amino acid sequence of SEQ ID NO: 90; (b) a light chain variable domain (VL)
comprising a
sequence having at least 95% sequence identity to the amino acid sequence of
SEQ ID NO: 71; or (c)
a VH as in (a) and a VL as in (b). In some embodiments, the antibody comprises
a VH comprising
the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody
comprises a VL
comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the
antibody
comprises the amino acid sequence of SEQ ID NO: 90 and a VL comprising the
amino acid sequence
of SEQ ID NO: 71.
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[0026] In some embodiments, the antibody comprises (a) a heavy chain
variable domain (VH)
comprising a sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID
NO: 91; (b) a light chain variable domain (VL) comprising a sequence having at
least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 72; or (c) a VH as in (a)
and a VL as in (b). In
some embodiments, the antibody comprises a VH comprising the amino acid
sequence of SEQ ID
NO: 91. In some embodiments, the antibody comprises a VL comprising the amino
acid sequence of
SEQ ID NO: 72. In some embodiments, the antibody comprises a VH comprising the
amino acid
sequence of SEQ ID NO: 91 and a VL comprising the amino acid sequence of SEQ
ID NO: 72.
[0027] In some embodiments, the antibody comprises a heavy chain comprising
the amino acid
sequence of SEQ ID NO: 106. In some embodiments, the antibody comprises a
light chain
comprising the amino acid sequence of SEQ ID NO: 114.
[0028] In some embodiments, the antibody comprises (a) a heavy chain
variable domain (VH)
comprising a sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID
NO: 92; (b) a light chain variable domain (VL) comprising a sequence having at
least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 73; or (c) a VH as in (a)
and a VL as in (b). In
some embodiments, the antibody comprises a VH comprising the amino acid
sequence of SEQ ID
NO: 92. In some embodiments, the antibody comprises a VL comprising the amino
acid sequence of
SEQ ID NO: 73. In some embodiments, the antibody comprises a VH comprising the
amino acid
sequence of SEQ ID NO: 92 and a VL comprising the amino acid sequence of SEQ
ID NO: 73.
[0029] In some embodiments, the antibody comprises a heavy chain comprising
the amino acid
sequence of SEQ ID NO: 107. In some embodiments, the antibody comprises the
antibody comprises
a light chain comprising the amino acid sequence of SEQ ID NO: 115.
[0030] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to a
fibronectin-like domain of MerTK comprises (a) an HVR-H1 comprising the amino
acid sequence of
SEQ ID NO: 27, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:
28 and (c) an
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29. In some
embodiments, the
antibody further comprises (a) an HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 25;
(b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) an
HVR-L3
comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the
antibody
comprises (a) a heavy chain variable domain (VH) comprising a sequence having
at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 93; (b) a light
chain variable domain
(VL) comprising a sequence having at least 95% sequence identity to the amino
acid sequence of SEQ
ID NO: 74; or (c) a VH as in (a) and a VL as in (b). In some embodiments, the
antibody comprises a
VH comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments,
the antibody
comprises a VL comprising the amino acid sequence of SEQ ID NO: 74. In some
embodiments, the
antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 93
and a VL
comprising the amino acid sequence of SEQ ID NO: 74.
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[0031] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to a
fibronectin-like domain of MerTK comprises (a) an HVR-H1 comprising the amino
acid sequence of
SEQ ID NO: 33, (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:
34 and (c) an
HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35. In some
embodiments, the
antibody further comprises (a) an HVR-L1 comprising the amino acid sequence of
SEQ ID NO: 30;
(b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 31; and (c) an
HVR-L3
comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the
antibody
comprises (a) a heavy chain variable domain (VH) comprising a sequence having
at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 94; (b) a light
chain variable domain
(VL) comprising a sequence having at least 95% sequence identity to the amino
acid sequence of SEQ
ID NO: 75; or (c) a VH as in (a) and a VL as in (b). In some embodiments, the
antibody comprises a
VH comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments,
the antibody
comprises a VL comprising the amino acid sequence of SEQ ID NO: 75. In some
embodiments, the
antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 94
and a VL
comprising the amino acid sequence of SEQ ID NO: 75.
[0032] In an exemplary embodiment, an anti-MerTK antibody of the present
disclosure binds to
an immuno globulin-like domain of MerTK.
[0033] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to an
immunoglobulin-like domain of MerTK comprises (a) an HVR-H1 comprising the
amino acid
sequence of SEQ ID NO: 38, (b) an HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 39,
and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40. In some
embodiments,
the antibody further comprises (a) an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:
36; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c)
an HVR-L3
comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the
antibody
comprises (a) a heavy chain variable domain (VH) comprising a sequence having
at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 95; (b) a light
chain variable domain
(VL) comprising a sequence having at least 95% sequence identity to the amino
acid sequence of SEQ
ID NO: 76; or (c) a VH as in (a) and a VL as in (b). In some embodiments, the
antibody comprises a
VH comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments,
the antibody
comprises a VL comprising the amino acid sequence of SEQ ID NO: 76. In some
embodiments, the
antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 95
and a VL
comprising the amino acid sequence of SEQ ID NO: 76.
[0034] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to an
immunoglobulin-like domain of MerTK comprises (a) an HVR-H1 comprising the
amino acid
sequence of SEQ ID NO: 44, (b) an HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 45,
and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 46. In some
embodiments,
the antibody further comprises (a) an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:
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41; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42; and (c)
an HVR-L3
comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the
antibody
comprises (a) a heavy chain variable domain (VH) comprising a sequence having
at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 96; (b) a light
chain variable domain
(VL) comprising a sequence having at least 95% sequence identity to the amino
acid sequence of SEQ
ID NO: 77; or (c) a VH as in (a) and a VL as in (b). In some embodiments, the
antibody comprises a
VH comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments,
the antibody
comprises a VL comprising the amino acid sequence of SEQ ID NO: 77. In some
embodiments, the
antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 96
and a VL
comprising the amino acid sequence of SEQ ID NO: 77.
[0035] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to an
immunoglobulin-like domain of MerTK comprises (a) an HVR-H1 comprising the
amino acid
sequence of SEQ ID NO: 50, (b) an HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 51,
and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 52. In some
embodiments,
the antibody further comprises (a) an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:
47; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48; and (c)
an HVR-L3
comprising the amino acid sequence of SEQ ID NO: 49. In some embodiments, the
antibody
comprises (a) a heavy chain variable domain (VH) comprising a sequence having
at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 97; (b) a light
chain variable domain
(VL) comprising a sequence having at least 95% sequence identity to the amino
acid sequence of SEQ
ID NO: 78; or (c) a VH as in (a) and a VL as in (b). In some embodiments, the
antibody comprises a
VH comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments,
the antibody
comprises a VL comprising the amino acid sequence of SEQ ID NO: 78. In some
embodiments, the
antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 97
and a VL
comprising the amino acid sequence of SEQ ID NO: 78.
[0036] In some embodiments, the antibody comprises (a) a heavy chain
variable domain (VH)
comprising a sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID
NO: 98; (b) a light chain variable domain (VL) comprising a sequence having at
least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 79; or (c) a VH as in (a)
and a VL as in (b). In
some embodiments, the antibody comprises a VH comprising the amino acid
sequence of SEQ ID
NO: 98. In some embodiments, the antibody comprises a VL comprising the amino
acid sequence of
SEQ ID NO: 79. In some embodiments, the antibody comprises a VH comprising the
amino acid
sequence of SEQ ID NO: 98 and a VL comprising the amino acid sequence of SEQ
ID NO: 79.
[0037] In some embodiments, the antibody comprises a heavy chain comprising
the amino acid
sequence of SEQ ID NO: 108. In some embodiments, the antibody comprises a
light chain comprising
the amino acid sequence of SEQ ID NO: 116.
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[0038] In some embodiments, the antibody comprises (a) a heavy chain
variable domain (VH)
comprising a sequence having at least 95% sequence identity to the amino acid
sequence of SEQ ID
NO: 99; (b) a light chain variable domain (VL) comprising a sequence having at
least 95% sequence
identity to the amino acid sequence of SEQ ID NO: 80; or (c) a VH as in (a)
and a VL as in (b). In
some embodiments, the antibody comprises a VH comprising the amino acid
sequence of SEQ ID
NO: 99. In some embodiments, the antibody comprises a VL comprising the amino
acid sequence of
SEQ ID NO: 80. In some embodiments, the antibody comprises a VH comprising the
amino acid
sequence of SEQ ID NO: 99 and a VL comprising the amino acid sequence of SEQ
ID NO: 80.
[0039] In some embodiments, the antibody comprises a heavy chain comprising
the amino acid
sequence of SEQ ID NO: 109. In some embodiments, the antibody comprises a
light chain
comprising the amino acid sequence of SEQ ID NO: 117.
[0040] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to an
immunoglobulin-like domain of MerTK comprises (a) an HVR-H1 comprising the
amino acid
sequence of SEQ ID NO: 56, (b) an HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 57,
and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 58. In some
embodiments,
the antibody further comprises (a) an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:
53; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 54; and (c)
an HVR-L3
comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the
antibody
comprises (a) a heavy chain variable domain (VH) comprising a sequence having
at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 100; (b) a light
chain variable domain
(VL) comprising a sequence having at least 95% sequence identity to the amino
acid sequence of SEQ
ID NO: 81; or (c) a VH as in (a) and a VL as in (b). In some embodiments, the
antibody comprises a
VH comprising the amino acid sequence of SEQ ID NO: 100. In some embodiments,
the antibody
comprises a VL comprising the amino acid sequence of SEQ ID NO: 81. In some
embodiments, the
antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 100
and a VL
comprising the amino acid sequence of SEQ ID NO: 81.
[0041] In one aspect, the present disclosure provides an anti-MerTK
antibody binding to an
immunoglobulin-like domain of MerTK comprises (a) an HVR-H1 comprising the
amino acid
sequence of SEQ ID NO: 62, (b) an HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 63,
and (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 64. In some
embodiments,
the antibody further comprises (a) an HVR-L1 comprising the amino acid
sequence of SEQ ID NO:
59; (b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60; and (c)
an HVR-L3
comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the
antibody
comprises (a) a heavy chain variable domain (VH) comprising a sequence having
at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 101; (b) a light
chain variable domain
(VL) comprising a sequence having at least 95% sequence identity to the amino
acid sequence of SEQ
ID NO: 82; or (c) a VH as in (a) and a VL as in (b). In some embodiments, the
antibody comprises a
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VH comprising the amino acid sequence of SEQ ID NO: 101. In some embodiments,
the antibody
comprises a VL comprising the amino acid sequence of SEQ ID NO: 82. In some
embodiments, the
antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 101
and a VL
comprising the amino acid sequence of SEQ ID NO: 82.
[0042] In some embodiments, an anti-MerTK antibody of the present
disclosure is a full length
IgGl, IgG2, IgG3, or IgG4 antibody. In certain embodiments, the antibody is a
full length IgG1
antibody. In certain embodiments, the antibody comprises a LALAPG mutation. In
some
embodiments, the antibody comprises Q2 and L4 residues in the light chain
variable region and 148,
G49, and K71 residues in the heavy chain variable region. In some embodiments,
the antibody
comprises L4 and F87 in the light chain variable region and V24, 148, G49, and
K71 in the heavy
chain variable region. In some embodiments, the antibody comprises L4 and P43
in the light chain
variable region and K71 in the heavy chain variable region. In some
embodiments, the antibody
comprises G49 and V78 residues in the heavy chain variable region.
[0043] In certain embodiments, the anti-MerTK antibodies provided herein
bind to human
MerTK with a dissociation constant (Kd) of < 100 nM at 25 C. In certain
embodiments, the anti-
MerTK antibodies provided herein bind to cyno MerTK with a dissociation
constant (Kd) of < 100
nM at 25 C. In certain embodiments, the anti-MerTK antibodies provided herein
bind to mouse
MerTK with a dissociation constant (Kd) of <10 nM at 25 C. In certain
embodiments, the anti-
MerTK antibodies provided herein bind to rat MerTK with a dissociation
constant (Kd) of < 10 nM at
25 C. In certain embodiments, the anti-MerTK antibodies provided herein bind
to human MerTK
with a dissociation constant (Kd) of < 10 nM, < 5 nM, or < 2 nM at 25 C.
[0044] In one aspect, the present disclosure provides isolated antibodies
that compete for binding
to MerTK with a reference antibody. Such reference antibodies include an
antibody comprising a VH
comprising the amino acid sequence of SEQ ID NO: 83 and a VL comprising the
amino acid
sequence of SEQ ID NO: 65; an antibody comprising a VH comprising the amino
acid sequence of
SEQ ID NO: 84 and a VL comprising the amino acid sequence of SEQ ID NO: 66; an
antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL
comprising the
amino acid sequence of SEQ ID NO: 67; an antibody comprising a VH comprising
the amino acid
sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ
ID NO: 68; an
antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 87
and a VL
comprising the amino acid sequence of SEQ ID NO: 69; an antibody comprising a
VH comprising the
amino acid sequence of SEQ ID NO: 88 and a VL comprising the amino acid
sequence of SEQ ID
NO: 70; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 89 and a
VL comprising the amino acid sequence of SEQ ID NO: 70; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid
sequence of SEQ ID
NO: 71; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 91 and a
VL comprising the amino acid sequence of SEQ ID NO: 72; an antibody comprising
a VH comprising
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the amino acid sequence of SEQ ID NO: 92 and a VL comprising the amino acid
sequence of SEQ ID
NO: 73; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 93 and a
VL comprising the amino acid sequence of SEQ ID NO: 74; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 94 and a VL comprising the amino acid
sequence of SEQ ID
NO: 75; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 95 and a
VL comprising the amino acid sequence of SEQ ID NO: 76; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 96 and a VL comprising the amino acid
sequence of SEQ ID
NO: 77; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 97 and a
VL comprising the amino acid sequence of SEQ ID NO: 78; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 98 and a VL comprising the amino acid
sequence of SEQ ID
NO: 79; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 99 and a
VL comprising the amino acid sequence of SEQ ID NO: 80; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 100 and a VL comprising the amino acid
sequence of SEQ
ID NO: 81; and an antibody comprising a VH comprising the amino acid sequence
of SEQ ID NO:
101 and a VL comprising the amino acid sequence of SEQ ID NO: 82. In some
embodiments, the
isolated antibody binds to human MerTK. In some embodiments, the reference
antibody is Y323.
[0045] In one
aspect, the present disclosure provides isolated antibodies that compete for
binding
to the same epitope on MerTK as a reference antibody. Such reference
antibodies include an antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO: 83 and a VL
comprising the
amino acid sequence of SEQ ID NO: 65; an antibody comprising a VH comprising
the amino acid
sequence of SEQ ID NO: 84 and a VL comprising the amino acid sequence of SEQ
ID NO: 66; an
antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 85
and a VL
comprising the amino acid sequence of SEQ ID NO: 67; an antibody comprising a
VH comprising the
amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid
sequence of SEQ ID
NO: 68; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 87 and a
VL comprising the amino acid sequence of SEQ ID NO: 69; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 88 and a VL comprising the amino acid
sequence of SEQ ID
NO: 70; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 89 and a
VL comprising the amino acid sequence of SEQ ID NO: 70; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid
sequence of SEQ ID
NO: 71; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 91 and a
VL comprising the amino acid sequence of SEQ ID NO: 72; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 92 and a VL comprising the amino acid
sequence of SEQ ID
NO: 73; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 93 and a
VL comprising the amino acid sequence of SEQ ID NO: 74; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 94 and a VL comprising the amino acid
sequence of SEQ ID
NO: 75; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 95 and a
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VL comprising the amino acid sequence of SEQ ID NO: 76; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 96 and a VL comprising the amino acid
sequence of SEQ ID
NO: 77; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 97 and a
VL comprising the amino acid sequence of SEQ ID NO: 78; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 98 and a VL comprising the amino acid
sequence of SEQ ID
NO: 79; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 99 and a
VL comprising the amino acid sequence of SEQ ID NO: 80; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 100 and a VL comprising the amino acid
sequence of SEQ
ID NO: 81; and an antibody comprising a VH comprising the amino acid sequence
of SEQ ID NO:
101 and a VL comprising the amino acid sequence of SEQ ID NO: 82. In some
embodiments, the
isolated antibody binds to human MerTK. In some embodiments, the reference
antibody is Y323.
[0046] In one aspect, the present disclosure provides an isolated antibody
that binds to MerTK,
wherein the antibody comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO:
102 and a light chain comprising the amino acid sequence of SEQ ID NO: 110. In
one aspect, the
present disclosure provides an isolated antibody that binds to MerTK, wherein
the antibody comprises
a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light
chain comprising
the amino acid sequence of SEQ ID NO: 111. In one aspect, the present
disclosure provides an
isolated antibody that binds to MerTK, wherein the antibody comprises a heavy
chain comprising the
amino acid sequence of SEQ ID NO: 104 and a light chain comprising the amino
acid sequence of
SEQ ID NO: 112. In another aspect, the present disclosure provides an isolated
antibody that binds to
MerTK, wherein the antibody comprises a heavy chain comprising the amino acid
sequence of SEQ
ID NO: 105 and a light chain comprising the amino acid sequence of SEQ ID NO:
113. In one aspect,
the present disclosure provides an isolated antibody that binds to MerTK,
wherein the antibody
comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 106
and a light chain
comprising the amino acid sequence of SEQ ID NO: 114. In one aspect, the
present disclosure
provides an isolated antibody that binds to MerTK, wherein the antibody
comprises a heavy chain
comprising the amino acid sequence of SEQ ID NO: 107 and a light chain
comprising the amino acid
sequence of SEQ ID NO: 115. In another aspect, the present disclosure provides
an isolated antibody
that binds to MerTK, wherein the antibody comprises a heavy chain comprising
the amino acid
sequence of SEQ ID NO: 108 and a light chain comprising the amino acid
sequence of SEQ ID NO:
116. In still another aspect, the present disclosure provides an isolated
antibody that binds to MerTK,
wherein the antibody comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO:
109 and a light chain comprising the amino acid sequence of SEQ ID NO: 117.
[0047] In some embodiments, an anti-MerTK of the present disclosure reduces
MerTK-mediated
clearance of apoptotic cells. In a specific embodiment, the anti-MerTK
antibody reduces MerTK-
mediated clearance of apoptotic cells by phagocytes. In certain embodiments,
the phagocytes are
macrophages. In a specific embodiment, the macrophages are tumor-associated
macrophages.In some
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embodiments, the clearance of apoptotic cells is reduced as measured in an
apoptotic cell clearance
assay at room temperature. In some embodiments, an anti-MerTK antibody of the
present disclosure
increases circulating tumor DNA (ctDNA) in blood or plasma. In some
embodiments, an anti-MerTK
antibody of the present disclosure increases cell-free DNA (cfDNA) in blood or
plasma.
[0048] In some embodiments, an anti-MerTK of the present disclosure is a
monoclonal antibody.
In certain embodiments, the anti-MerTK antibody is a humanized or chimeric
antibody. In certain
embodiments, the anti-MerTK antibody is a human, humanized, or chimeric
antibody. In certain
embodiments, the anti-MerTK antibody is an antibody fragment that binds MerTK.
In certain
embodiments, the anti-MerTK antibody binds to a fibronectin-like domain or an
immunoglobulin-like
domain of MerTK. In certain embodiments, the anti-MerTK antibody binds to the
fibronectin-like
domain of MerTK. In certain embodiments, the anti-MerTK antibody binds to an
immuno globulin-
like domain of MerTK.
[0049] In one aspect, the present disclosure provides an isolated nucleic
acid that encodes any of
the anti-MerTK antibodies described herein. In another aspect, the present
disclosure provides a
vector including the nucleic acid encoding any of the anti-MerTK antibodies
described herein. In a
still further aspect, the present disclosure provides a host cell containing
the vector suitable for
expression of the nucleic acid encoding any of the anti-MerTK antibodies
described herein.
[0050] Further provided herein is a method of producing an anti-MerTK
antibody of the present
disclosure including culturing a host cell containing a nucleic acid that
encodes an anti-MerTK
antibody under conditions suitable for expression of the antibody. In some
embodiments, the method
further includes recovering the anti-MerTK antibody from the cell culture.
[0051] In one aspect, the present disclosure pertains to an immunoconjugate
including an anti-
MerTK antibody provided herein conjugated to a cytotoxic agent. In another
aspect, the present
disclosure pertains to a pharmaceutical formulation including any of the above
described anti-MerTK
antibodies and a pharmaceutically-acceptable carrier. In another aspect, the
present disclosure
pertains to a pharmaceutical formulation including any of the above described
anti-MerTK
immunoconjugates and a pharmaceutically-acceptable carrier.
[0052] In one aspect, the present disclosure provides the anti-MerTK
antibodies or
immunoconjugates as described above for use as a medicament. In some
embodiments, the use is in
treating cancer. In some embodiments, the use is in reducing MerTK-mediated
clearance of apoptotic
cells.
[0053] Further provided herein are uses of the anti-MerTK antibodies or
immunoconjugates as
described above in the manufacture of a medicament. In some embodiments, the
medicament is for
treatment of cancer. In some embodiments, the cancer expresses functional
STING, functional Cx43,
and functional cGAS polypeptides. In some embodiments, the cancer comprises
tumor-associated
macrophages that express functional STING polypeptides. In some embodiments,
the cancer
comprises tumor cells that express functional cGAS polypeptides. In some
embodiments, the cancer
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comprises tumor cells that express functional Cx43 polypeptides. In certain
embodiments, the cancer
is colon cancer. In some embodiments, the medicament is for reducing MerTK-
mediated clearance of
apoptotic cells.
[0054] In some embodiments, the uses may further include an additional
therapy or
administration of an effective amount of an additional therapeutic agent. In
some embodiments, the
additional therapy is selected from one or more of tamoxifen, letrozole,
exemestane, anastrozole,
irinotecan, cetuximab, fulvestrant, vinorelbine, erlotinib, bevacizumab,
vincristine, imatinib mesylate,
sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine, methotrexate,
vinblastine, carboplatin,
paclitaxel, 5-fluorouracil, doxorubicin, bortezomib, melphalan, prednisone,
and docetaxel. In some
embodiments, the additional therapeutic agent is an immune checkpoint
inhibitor. In some
embodiments, the immune checkpoint inhibitor is selected from one or more of a
cytotoxic T-
lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death
protein 1 (PD-1)
binding antagonist, or a programmed death-ligand 1 (PDL1) binding antagonist.
In some
embodiments, the immune checkpoint inhibitor is a PDL1 binding antagonist. In
an exemplary
embodiment, the PDL1 binding antagonist is an anti-PDL1 antibody. In some such
embodiments, the
anti-PDL1 antibody is atezolizumab. In some embodiments, the medicament is
further used in
combination with an effective amount of a chemotherapeutic agent.
[0055] In another aspect, provided herein are methods for treating or
delaying progression of
cancer in an individual including administering to the individual an effective
amount of an anti-
MerTK antibody or an immunoconjugate thereof as described in the present
disclosure. In some
embodiments, the cancer expresses functional STING, functional Cx43, and
functional cGAS
polypeptides. In some embodiments, the cancer comprises tumor-associated
macrophages that
express functional STING polypeptides. In some embodiments, the cancer
comprises tumor cells that
express functional cGAS polypeptides. In some embodiments, the cancer
comprises tumor cells that
express functional Cx43 polypeptides. In certain embodiments, the cancer is
colon cancer.
[0056] In some embodiments, the methods may further include an additional
therapy or
administration of an effective amount of an additional therapeutic agent. In
some embodiments, the
additional therapy is selected from one or more of tamoxifen, letrozole,
exemestane, anastrozole,
irinotecan, cetuximab, fulvestrant, vinorelbine, erlotinib, bevacizumab,
vincristine, imatinib mesylate,
sorafenib, lapatinib, trastuzumab, cisplatin, gemcitabine, methotrexate,
vinblastine, carboplatin,
paclitaxel, 5-fluorouracil, doxorubicin, bortezomib, melphalan, prednisone,
and docetaxel.
[0057] In some embodiments, the additional therapeutic agent is an immune
checkpoint
inhibitor. In some embodiments, the immune checkpoint inhibitor is selected
from one or more of a
cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed
cell death protein 1
(PD-1) binding antagonist, or a programmed death-ligand 1 (PDL1) binding
antagonist. In some
embodiments, the immune checkpoint inhibitor is a PDL1 binding antagonist. In
an exemplary
embodiment, the PDL1 binding antagonist is an anti-PDL1 antibody. In some such
embodiments, the
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anti-PDL1 antibody is atezolizumab. In some embodiments, the methods may
further comprise
administering an effective amount of an additional chemotherapeutic agent to
the individual.
[0058] In another aspect, provided herein are methods of reducing MerTK-
mediated clearance of
apoptotic cells in an individual including administering to the individual an
effective amount of an
anti-MerTK antibody or an immunoconjugate thereof as described in the present
disclosure to reduce
MerTK-mediated clearance of apoptotic cells. In some embodiments, the
clearance of apoptotic cells
is reduced by about 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, 2-10, 2-8, 2-5, 2-4, 2-3, 3-
10, 3-8, 3-5, or 3-4 fold or
by about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,
2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,
3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,
4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,
5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,
7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
7.8, 7.9, or 8.0 fold.
[0059] It is to be understood that one, some, or all of the properties of
the various embodiments
described herein may be combined to form other embodiments of the present
disclosure. These and
other aspects of the disclosure will become apparent to one of skill in the
art. These and other
embodiments of the disclosure are further described by the detailed
description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIGS. 1A and 1B: The light chain and heavy chain variable regions of
each MerTK
specific antibody generated in rabbits were amplified by PCR and cloned into
expression vectors for
purification and sequencing The amino acid sequences for the light chain
variable region (FIG.1A)
and heavy chain variable region (FIG. 1B) were aligned. Residue numbers
referenced are matched to
the sequence defined in Kabat et al. and the CDR sequences are underlined. SEQ
ID NOs are as
follows: Rbt8F4 (SEQ ID NO: 65), Rbt9E3.FN (SEQ ID NO: 66), Rbt10C3 (SEQ ID
NO: 69),
Rbt10F7 (SEQ ID NO: 71), Rbt11G11 (SEQ ID NO: 76), Rbt12H4 (SEQ ID NO: 77),
Rbt13B4 (SEQ
ID NO: 78), Rbt13D8 (SEQ ID NO: 74), Rbt14C9 (SEQ ID NO: 81), Rbt18G7 (SEQ ID
NO: 82), and
Rbt22C4 (SEQ ID NO: 75). SEQ ID NO in FIG. 1B are as follows: Rbt8F4 (SEQ ID
NO: 83),
Rbt9E3.FN (SEQ ID NO: 84), Rbt10C3 (SEQ ID NO: 87), Rbtl OF7 (SEQ ID NO: 90),
Rbt11G11
(SEQ ID NO: 95), Rbt12H4 (SEQ ID NO: 96), Rbt13B4 (SEQ ID NO: 97), Rbt13D8
(SEQ ID NO:
93), Rbt14C9 (SEQ ID NO: 100), Rbt18G7 (SEQ ID NO: 101), and Rbt22C4 (SEQ ID
NO: 94).
[0061] FIGS. 2A, 2B, 2C & 2D: Antibodies 10F7, 9E3, 13B4, and 10C3 were
selected for
humanization. The amino acid sequences of the light chain and heavy chain
variable regions for
antibody 10F7 before humanization, following phase 1 of humanization (.v1),
and following phase 2
of humanization (.v16) were aligned (FIG. 2A). The amino acid sequences of
light chain and heavy
chain variable regions for antibody 9E3 before humanization, following phase 1
of humanization
(.v1), and following phase 2 of humanization (.v16) were aligned (FIG. 2B).
The amino acid
sequences of light chain and heavy chain variable regions for antibody 13B4
before humanization,
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following phase 1 of humanization (.v1), and following phase 2 of humanization
(.v16) were aligned
(FIG. 2C). The amino acid sequences of light chain and heavy chain variable
regions for antibody
10C3 before humanization, following phase 1 of humanization (.v1), and
following phase 2 of
humanization (.v14) were aligned (FIG. 2D). Residue numbers referenced are
matched to the
sequence defined in Kabat et al. and the CDR sequences are underlined. SEQ ID
NOs for light chain
sequences are as follows: Rbtl OF7 (SEQ ID NO: 71), h10F7.v1 (SEQ ID NO: 72),
hl OF7.v16 (SEQ
ID NO: 73), Rbt9E3.FN (SEQ ID NO: 66), h9E3.FN.v1 (SEQ ID NO: 67), h9E3.FN.v16
(SEQ ID
NO: 68), Rbt13B4 (SEQ ID NO: 78), h13B4.v1 (SEQ ID NO: 79), h13B4.v16 (SEQ ID
NO: 80),
Rbt10C3 (SEQ ID NO: 69), h10C3.v1 and h10C3.v14 (SEQ ID NO: 70). SEQ ID NO for
heavy chain
sequences in FIG.2A-2D are as follows: Rbtl OF7 (SEQ ID NO: 90), h10F7.v1 (SEQ
ID NO: 91),
hl OF7.v16 (SEQ ID NO: 92), Rbt9E3.FN (SEQ ID NO: 84), h9E3.FN.v1 (SEQ ID NO:
85),
h9E3.FN.v16 (SEQ ID NO: 86), Rbt13B4 (SEQ ID NO: 97), h13B4.v1 (SEQ ID NO:
98), h13B4.v16
(SEQ ID NO: 99), Rbt10C3 (SEQ ID NO: 87), h10C3.v1 (SEQ ID NO: 88), and
h10C3.v14 (SEQ ID
NO: 89).
100621 FIG. 3: Epitope binning was used to determine epitope domain
specificity for each anti-
MerTK antibody. Antibodies 8F4, 22C4, and 13D8, raised against mouse MerTK,
and antibodies
10C3, 9E3.FN, 10F7, 22C4, 8F4, and 13D8, raised against human MerTK, competed
for binding with
each other. Antibodies 12H4, 18G7, 14C9, and 11G11, raised against mouse
MerTK, and antibodies
13B4, 12H4, 18G7, and 11G11, raised against human MerTK, competed with each
other. As
described further in the Examples below, antibodies 10C3, 9E3.FN, 10F7, 22C4,
8F4, and 13D8 bind
to MerTK's fibronectin-like domain, and antibodies 13B4, 12H4, 18G7, and 11G11
bind to MerTK's
Ig-like domain.
[0063] FIGS. 4A, 4B, 4C, 4D & 4E: Efferocytosis assays were carried out to
evaluate the in
vitro phagocytosis inhibiting activity of anti-MerTK antibodies. Anti-MerTK
antibodies inhibited the
phagocytic activity of human macrophages isolated from three different donors
(FIGS. 4A-4C). Anti-
MerTK antibody h13B4.v16 (13B4 Fully Humanized), an Ig-domain binding
antibody, was 5.2x more
potent at inhibiting human macrophage phagocytosis compared to anti-MerTK
antibody h10F7.v16
(10F7 Fully Humanized), a fibronectin-domain binding antibody (FIG. 4D). Anti-
MerTK antibody
14C9 mIgG2a LALAPG, was 4.8x more potent at inhibiting mouse macrophage
phagocytosis
compared to anti-MerTK antibody hl 0F7.v16 (10F7 Fully Humanized) (FIG. 4E).
[0064] FIGS. 5A, 5B & 5C: An apoptotic cell clearance assay was carried out
to evaluate the in
vivo activity of anti-MerTK antibodies. Apoptotic cells accumulated 8 hours
after Dex treatment and
were mostly cleared by 24 hours (FIG. 5A). Anti-MerTK (clone 14C9, mIgG2a,
LALAPG) but not
the control antibody anti-gp120 (mIgG2a, LALAPG) blocked the clearance of
apoptotic cells in the
thymus 24 hours after Dex treatment (FIG. 5B). Anti-MerTK antibodies blocked
the clearance of
apoptotic cells relative to the anti-gp120 control (FIG. 5C).
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[0065] FIGS. 6A, 6B, 6C & 6D: Tumor efficacy studies were carried out in
the MC-38
syngeneic tumor model to determine whether anti-MerTK antibodies affect tumor
growth. Changes in
individual tumor size (FIGS. 6A & 6B; each line represents a single tumor) and
mean tumor size
(FIGS. 6C & 6D), were measured over time for each treatment group. In the
tumor volume tracking
plots, gray lines represent the tumor size of animals that were still in the
study as of the date of data
collection (FIGS. 6A & 6B). Red lines represent animals with ulcerated or
progressed tumors that
were euthanized and removed from study (FIGS. 6A & 6B). Red horizontal dashed
lines indicate a
doubling in tumor volume from the start of treatment while green horizontal
dashed lines represent the
smallest measureable tumor volume (FIGS. 6A & 6B). Animals with tumors in the
area below the
green dashed line are considered to have had a complete response. The
treatment combination of anti-
gp120 and anti-PDL1 antibodies did not inhibit tumor growth to a large degree.
However, treatments
combining anti-PDL1 and anti-MerTK antibodies exhibited enhanced anti-tumor
activity (FIGS. 6A-
6D).
[0066] FIGS. 7A, 7B & 7C: A schematic depiction of blocking MerTK-dependent
efferocytosis
by anti-MerTK antibody (FIG. 7A). An in vitro efferocytosis assay was carried
out to evaluate the
phagocytosis inhibiting effect of anti-MerTK 14C9 (mIgG2a LALAPG) antibody
treatment.
Peritoneal macrophages (green) treated with anti-MerTK 14C9 (mIgG2a LALAPG)
antibody
exhibited approximately 8x less phagocytic clearance of apoptotic thymocytes
(red) as compared to
macrophages treated with control antibody anti-gp120 (mIgG2a LALAPG) (black)
(FIG. 7B). An in
vivo apoptotic cell clearance assay was carried out to determine the effect of
anti-MerTK treatment on
the clearance of apoptotic cells in the thymus. At 24 hours following
induction of thymocyte
apoptosis with dexamethasone (Dex), mice treated with anti-MerTK 14C9 (mIgG2a
LALAPG)
antibody accumulated approximately 6x more apoptotic thymus cells (red) as
compared to mice
treated with control antibody anti-gp120 (mIgG2a LALAPG) (black) (FIG. 7C).
[0067] FIGS. 8A, 8B, 8C, 8D & 8E: An in vitro assay to quantify the effect
of anti-MerTK
14C9 (mIgG2a LALAPG) antibody treatment on ligand-mediated MerTK signaling was
performed by
measuring pAKT levels in macrophages incubated with the ligand hGAS6-Fc (EC50
= ¨ 84 pM).
Treatment of J774A.1 macrophages with increasing concentrations of anti-MerTK
14C9 (mIgG2a
LALAPG) antibody blocked ligand-mediated MerTK signaling, as evidenced by
lower levels of
pAKT in macrophages treated with anti-MerTK 14C9 (mIgG2a LALAPG) as compared
to
macrophages treated with the isotype control antibody (FIG. 8A). An apoptotic
cell clearance assay
was carried out to evaluate the in vivo effect of Dex on thymocytes. Apoptotic
thymocytes
accumulated 8 hours after Dex treatment and were mostly cleared by 24 hours
(FIG. 8B). The
distribution of the MerTK protein within MC38 tumor sections was imaged using
fluorescence
microscopy. MerTK protein co-localized with CD68, a marker of tumor-associated
macrophages
(TAMs), indicating that MerTK is specifically expressed in TAMs (FIG. 8C). No
background signal
in Mertk-1- tissue sections stained with anti-MerTK 14C9 (mIgG2a LALAPG)
antibody was observed.
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(FIG. 8C). The distribution of MerTK expression was determined using
expression data from The
Cancer Genome Atlas (TCGA). MerTK expression exhibited greater correlation
with the abundance
of TAMs compared to other immune cell types (FIG. 8D). An efferocytosis assay
was carried out to
evaluate the inhibiting effect of anti-MerTK 14C9 (mIgG2a LALAPG) antibody on
in vitro
phagocytosis of apoptotic thymocytes (AC, red) by TAMs (TAM, green). Anti-
MerTK 14C9
(mIgG2a LALAPG) antibody inhibited the phagocytic activity of TAMs isolated
from MC38 tumors
(FIG. 8E).
[0068] FIGS. 9A, 9B, 9C, 9D & 9E: An RNA-sequencing experiment to evaluate
the effect of
anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment on the gene expression
pattern of MC38
TAMs. Anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment caused significant
changes to gene
expression in TAMs (FIG. 9A). A Gene Set Enrichment Analysis (GSEA) was
carried out to uncover
gene groups that were differentially regulated in response to treatment with
anti-MerTK 14C9
(mIgG2a LALAPG) antibody. The IFN-alpha response gene group was enriched
following anti-
MerTK 14C9 (mIgG2a LALAPG) antibody treatment (FIG. 9B). The effect of anti-
MerTK 14C9
(mIgG2a LALAPG) antibody treatment on the expression of Ifnb 1 and multiple
interferon stimulated
genes (ISGs) in TAMs was evaluated by qPCR. The indicated genes were more
highly expressed
following anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment relative to
control antibody
treatment (FIG. 9C). A quantitative ELISA was carried out to determine the
effect of anti-MerTK
14C9 (mIgG2a LALAPG) antibody treatment on IFN-beta protein levels. Anti-MerTK
14C9 (mIgG2a
LALAPG) antibody treatment led to a significant accumulation of IFN-beta
protein in MC38 tumors
(FIG. 9D). The effect of anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment on
IFN-beta
expression in the indicated MC38 tumor-derived cell types was evaluated by
qPCR. IFN-beta was
more highly expressed in CD45+ cells and TAMs treated with anti-MerTK 14C9
(mIgG2a LALAPG)
relative to cells treated with the control antibody. No significant changes in
IFN-beta expression were
observed in CD45- cells or dendritic cells (DC) (FIG. 9E).
[0069] FIGS. 10A, 10B, 10C, 10D & 10E: A method to isolate TAMs from tumor-
derived
single cell suspensions is depicted (FIG. 10A). The purity of isolated TAMs
was evaluated by FACS
(FIG. 10B). Statistical analysis, depicted as a Volcano plot, identified genes
whose expression was
increased, decreased, or unchanged by anti-MerTK 14C9 (mIgG2a LALAPG) antibody
treatment
(FIG. 10C). A Gene Set Enrichment Analysis (GSEA) was carried out to uncover
gene groups that
were differentially regulated in response to treatment with anti-MerTK 14C9
(mIgG2a LALAPG)
antibody. The indicated gene groups are ranked according to their degree of
enrichment following
anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment (FIG. 10D). qPCR analysis
was undertaken
to quantify the effect of anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment
on the expression
of the indicated ISGs in total MC38 tumors. The indicated genes were more
highly expressed
following anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment relative to a
control antibody
(FIG. 10E).
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[0070] FIGS. 11A & 11B: qPCR analysis was carried out to quantify the
effect of anti-MerTK
14C9 (mIgG2a LALAPG) antibody treatment on the expression of the indicated
genes in MC38
tumor-derived TAMS (FIG. 11A) or total MC38 tumor homogenate (FIG. 11B). Actb,
Gapdh,
Rp113a, Rp119, Hprt, and Rp14 were used as housekeeping genes.
[0071] FIGS. 12A, 12B & 12C: An in vivo antigen presentation assay was
employed to evaluate
the effect of anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment on antigen
presentation by
TAMs and dendritic cells (DCs). Anti-MerTK 14C9 (mIgG2a LALAPG) antibody
treatment
significantly increased the presentation of the MC38.0VA tumor-derived
SIINFEKL antigen bound
to the MHC Class I molecule, H-2K' by TAMs but not DCs (FIG 12A). The
expression of CD86, a
protein that promotes T cell activation, was quantified to evaluate the effect
of the anti-MerTK 14C9
(mIgG2a LALAPG) antibody on T cell activation. Anti-MerTK 14C9 (mIgG2a LALAPG)
antibody
treatment induced higher levels of CD86 on TAMs but not on DCs (FIG 12A). The
effect of anti-
MerTK 14C9 (mIgG2a LALAPG) treatment on productive rearrangements and
clonality of T cell
receptors (TCR) was measured by genomic DNA sequencing of MC38 tumor-derived T
cells. Anti-
MerTK 14C9 (mIgG2a LALAPG) antibody treatment led to significantly more TCR
clonality and
productive rearrangements relative to a control antibody (FIG. 12B). The
relative abundance of
CD8+, CD4+ and pl5e tetramer-reactive T cells in MC38 tumors was quantified to
determine the
effect of anti-MerTK 14C9 (mIgG2a LALAPG) treatment on antitumor immune
response. Anti-
MerTK 14C9 (mIgG2a LALAPG) treatment significantly enhanced the antitumor
response relative to
a control antibody, as evidenced by significant increases in the relative
abundance of CD8+ and pl5e
tetramer-reactive T cells following anti-MerTK antibody treatment (FIG. 12C).
[0072] FIGS. 13A, 13B & 13C: The protein levels of CCL3, CCL4, CCL5, CCL7
and CCL12
were quantified in tumor homogenates to evaluate the effect of anti-MerTK 14C9
(mIgG2a LALAPG)
treatment on autocrine and paracrine cytokines and chemokines. Anti-MerTK 14C9
(mIgG2a
LALAPG) antibody treatment caused a significant enrichment of all tested
proteins relative to
treatment with a control antibody (FIG. 13A). The expression of ISGs was
determined by qPCR
analysis in peripheral blood mononuclear cells (PBMCs) to determine the effect
of anti-MerTK 14C9
(mIgG2a LALAPG) treatment. No significant difference in the expression of the
indicated genes was
observed following anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment relative
to a control
antibody (FIG. 13B). Quantification of the expression of the indicated
cytokines and chemokines in
tumors (n = 10) revealed no significant effect of anti-MerTK 14C9 (mIgG2a
LALAPG) treatment.
[0073] FIGS. 14A, 14B & 14C: Gating strategies as depicted in the
representative FACS plots
in FIG. 14A were employed to isolate specific cell types from single cell
suspensions of MC38 tumors
(FIG. 14A). The frequency of TAMs and DCs over time in MC38 tumors (n = 8, day
8; n = 10, days
13 and 15) was quantified. TAMs were considerably more abundant than DCs in
MC38 tumors and
the frequency of CD45+ TAMs increased in tumors over time while DCs remain
constant (FIG. 14B).
To evaluate the effects of anti-MerTK 14C9 (mIgG2a LALAPG) treatment on CD206
expression in
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TAMs, flow cytometric analysis was carried out and the MFI, median
fluorescence intensity (n = 10)
was reported. Anti-MerTK 14C9 (mIgG2a LALAPG) antibody treatment caused a
decrease of CD206
expression on TAMs (FIG. 14C).
[0074] FIGS. 15A, 15B & 15C: MC38 tumors were treated either with single
agent anti-MerTK
14C9 (mIgG2a LALAPG) treatment started at early progression stage (FIG. 15A)
or combination
treatment with anti-MerTK 14C9 (mIgG2a LALAPG) and anti-PD-Li at established
stage (n = 10)
(FIG. 15B). Single agent anti-MerTK 14C9 (mIgG2a LALAPG) treatment inhibited
the growth of
early progression stage tumors (FIG. 15A). Combination treatment with anti-PD-
Li and anti-MerTK
14C9 (mIgG2a LALAPG) antibody at established stage inhibited the growth of
MC38 tumors, while
single agent anti-MerTK 14C9 (mIgG2a LALAPG) or anti-PD-Li treatment had
marginal or modest
effects, respectively (FIG. 15B). Established MC38 tumors were treated with
anti-MerTK 14C9
(mIgG2a LALAPG) in combination with gemcitabine (Gem) and anti-PD-1 (n =15,
control Ab group;
n = 8, anti-PD-1 + anti-MerTK 14C9 (mIgG2a LALAPG); n = 10, other groups).
Anti-MerTK 14C9
(mIgG2a LALAPG) treatment in combination with gemcitabine (Gem) and anti-PD-1
inhibited tumor
growth. Single agent anti-PD-1 or Gem therapy inhibited tumor growth to a
lesser extent than anti-
PD-1 and/or Gem combination treatments with anti-MerTK 14C9 (mIgG2a LALAPG)
(FIG. 15C).
Both individual tumor growth curves and LME-fitted tumor growth curves of each
group are
presented (FIGS. 15A, 15B & 15C).
[0075] FIGS. 16A & 16B: The expression of representative ISGs in tumors
treated with anti-
MerTK 14C9 (mIgG2a LALAPG) in the presence or absence of anti-IFNAR1 (n = 5)
was quantified
to evaluate the effect of Type 1 IFN signaling on anti-MerTK 14C9 (mIgG2a
LALAPG) treatment.
Anti-IFNAR1 treatment abolished the enhanced expression of the indicated ISGs
caused by anti-
MerTK 14C9 (mIgG2a LALAPG) (FIG. 16A). The growth of MC38 tumors treated with
a
combination of anti-MerTK 14C9 (mIgG2a LALAPG) and anti-PD-Li along with anti-
IFNARI was
evaluated to determine the effect of Type 1 IFN signaling on combination anti-
MerTK 14C9
(mIgG2a LALAPG) and anti-PD-Li treatment (n=10). Anti-IFNAR1 antibody
treatment reduced the
tumor-inhibiting effect of combination anti-MerTK 14C9 (mIgG2a LALAPG) and
anti-PD-Li
therapy. Both individual tumor growth curves and LME-fitted tumor growth
curves of each group are
presented (FIG. 16B).
[0076] FIGS. 17A, 17B & 17C: The growth of MC38 tumors treated with single
agent anti-
MerTK 14C9 (mIgG2a LALAPG) therapy along with anti-IFNARI antibody was
evaluated (n=10) to
determine the effect of Type 1 IFN signaling on anti-MerTK 14C9 (mIgG2a
LALAPG) treatment.
Anti-IFNAR1 antibody treatment negated the tumor-inhibiting effect of anti-
MerTK 14C9 (mIgG2a
LALAPG) antibody therapy (FIG. 17A). The expression of representative ISGs in
MC38 tumors
growing in WT or Stinggt/gt mice was quantified to evaluate the effect of
STING on anti-MerTK 14C9
(mIgG2a LALAPG) antibody treatment (n = 9, WT host; n =10, Stinggtigt host).
STING disruption
abolished the enhanced expression of the indicated ISGs caused by anti-MerTK
14C9 (mIgG2a
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LALAPG) (FIG. 17B). The growth of MC38 tumors in WT or Stinggt/gt host mice
was quantified to
evaluate the effect of STING on anti-MerTK 14C9 (mIgG2a LALAPG) antibody
treatment (n=10).
STING disruption abolished the tumor-inhibiting effect of anti-MerTK 14C9
(mIgG2a LALAPG)
treatment (FIG. 17C). Both individual tumor growth curves and LME-fitted tumor
growth curves of
each group are presented (FIGS. 17A & 17C).
[0077] FIGS. 18A, 18B, 18C, 18D, 18E & 18F: Cytoplasmic DNA transfection
experiments
were carried out to evaluate the functions of STING and cGAS in the response
to cytoplasmic DNA in
macrophages. Accumulation of IFN-beta required both functional STING (FIG.
18A) and cGAS
(FIG. 18B) expression in macrophages in response to DNA transfection. Western
blot analysis of
cGAS and STING expression in MC38 tumor cells and J774A.1 macrophages
determined that
J774A.1 macrophages express cGAS and STING, while MC38 tumor cells only
express cGAS (FIG.
18C).The expression of representative ISGs in WT and cGAS-/- AB22 tumors was
quantified to
evaluate the role of cGAS expression in tumor cells during anti-MerTK
treatment. Disruption of
cGAS expression in tumor cells abolished the accumulation of the indicated
ISGs in response to anti-
MerTK treatment (FIG. 18D). The growth of WT or cGAS-/- MC38 tumors was
quantified to evaluate
the effect of cGAS expression in tumor cells on anti-MerTK as a single agent
or in combination with
anti-PD-Li (n = 9, WT MC38 with combination treatment; n = 10, other groups).
Tumor growth
inhibition by anti-MerTK and anti-PD-Li combination therapy was reduced in
cGAS-/- MC38 tumors.
Both individual tumor growth curves and LME-fitted tumor growth curves of each
group are
presented (FIG. 18E). Protein quantification by LC-MS/MS was used to measure
cGAMP production
in MC38 tumor cells, which increased in WT tumor cells following transfection
with HT-DNA, but
not in cGAS-/- tumor cells (FIG. 18F).
[0078] FIGS. 19A, 19B, 19C, 19D, & 19E: The production of IFN-beta protein
from WT and
Stinggt/gtBMDMs (FIG. 19A) or WT and cGAS-/- J774A.1 macrophages (FIG. 19B)
cocultured with
UV-irradiated WT or cGAS-/- MC38 cells was quantified. IFN-beta protein
accumulation was
dependent on cGAS expression in tumor cells and STING expression in
macrophages (FIGS. 19A &
19B). cGAS expression in macrophages was dispensable for IFN-beta protein
accumulation (FIG.
19B). The expression of representative ISGs in WT or cGAS-/- MC38 tumors
growing in WT host mice
(n = 10) was quantified to evaluate the effect of cGAS expression in tumor
cells on anti-MerTK single
agent therapy. cGAS disruption in tumor cells abolished the enhanced
expression of the indicated
ISGs in response to anti-MerTK treatment (FIG. 19C). The growth of WT or cGAS-
/- early stage
MC38 tumors grown in WT host mice was measured following single agent anti-
MerTK or anti-PD-
Li treatments (n = 10). cGAS deficient tumor cells were resistant to single
agent anti-MerTK or anti-
PD-Lltreatments, as measured by tumor growth inhibition. Both individual tumor
growth curves and
LME-fitted tumor growth curves of each group are presented (FIGS. 19D & 19E).
[0079] FIGS. 20A, 20B, 20C, 20D & 20E: Western blot analysis was carried
out to confirm loss
of Cx43 protein in Cx4.3-/- MC38 tumor cells (FIG. 20A). A schematic diagram
of a dye transfer assay
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measuring calcein movement between cells through Cx43 is depicted (FIG. 20B).
The dye transfer
assay of FIG. 20B was carried out to quantify the role of Cx43 in calcein
transfer between MC38
tumor cells (FIG. 20C) or from macrophages to tumor cells (FIG. 20D). Loss of
Cx43 compromised
the transfer of fluorescent dye calcein between MC38 cells (FIG. 20C) and from
J774A.1
macrophages to MC38 tumor cells (FIG. 20D). The growth of WT or Cx43 -/- MC38
tumors in WT
host mice was measured following treatment with anti-MerTK and anti-PD-Li
combination therapy
(n = 10, WT MC38; n = 8, Cx43 -/- MC38). Cx43 deficient tumor cells were
resistant to anti-MerTK
and anti-PD-Li combination therapy, as measured by tumor growth inhibition.
Both individual tumor
growth curves and LME-fitted tumor growth curves of each group are presented
(FIG. 20E).
[0080] FIGS. 21A, 21B, 21C & 21D: Schematic diagram of gap junction-
dependent transfer of
cGAMP from MC38 cells, and production of IFN-beta by macrophages (FIG. 21A).
The production
of IFN-beta protein from J774A.1 macrophages in coculture with HT-DNA
transfected (+ DNA) WT
or Cx43 -/- MC38 tumor cells was quantified. Disruption of Cx43 in tumor cells
abolished the
increased production of IFN-beta by macrophages caused by DNA transfection of
tumor cells (FIG.
21B). The mRNA expression of representative ISGs in Cx43 -/- MC38 tumors was
quantified to
determine the effect of Cx43 disruption in tumor cells on anti-MerTK treatment
(n = 10, control Ab;
n = 9, anti-MerTK). Treatment of Cx43 -/- MC38 tumors with anti-MerTK led to
no significant changes
in the expression of ISGs (FIG. 21C). A model depicting blockade of MerTK-
dependent innate
immune checkpoint. MerTK signaling in TAMs mediates rapid clearance of
stressed or dying tumor
cells, resulting in quiescent disposal of tumor-derived materials without
alerting the immune system.
Treatment with anti-MerTK prevents efferocytosis, allowing prolonged
production of cGAMP by
cGAS-expressing tumor cells and increased transfer of cGAMP via gap junctions
to host
macrophages. IFN-beta produced by TAMs acts in an autocrine/paracrine manner
to increase antigen
presentation and expression of co-stimulatory molecules by antigen presenting
cells, ultimately
leading to enhanced T cell response (FIG. 21D).
[0081] FIGS. 22A & 22B: Quantification of circulating tumor DNA (ctDNA) and
cell-free DNA
(cfDNA) in a mouse MC38 tumor model upon treatment with anti-MerTK or control
antibody. MC38
tumor cells were inoculated into C57BL/6J mice. Anti-MerTK or control antibody
was administered
after tumors were established. Three days after anti-MerTK treatment, a
significant increase of ctDNA
in the plasma of tumor-bearing mice was detected (FIG. 22A). Anti-MerTK also
increased the level of
host-derived cfDNA in blood circulation (FIG. 22B). Indicated p-values are
based on unpaired, two-
tailed Student's t-test. These results demonstrate that in tumor
microenvironment anti-MerTK
treatment was able to block the ongoing clearance of apoptotic cells by TAMs.
[0082] FIG. 23 shows the analysis of anti-MerTK antibody binding affinity
to human MerTK
using surface plasmon resonance (SPR). Binding affinity of 10 commercial
antibodies and
h13B4.v16 to human MerTK was determined. Binding affinities were observed as
follows: 0.4nM for
Y323, 6.8nM for A3KCAT, 7.6nM for 590H11G1E3, 17.3nM for MAB8912-100 and 1.6nM
for
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h13B4.v16. The remaining six antibodies (i 0g86D21F1 I, 2D2, 7E5G1, 7N-20,
MAB891, and
MAB8911) showed no binding to human MerTK.
[0083] FIGS. 24A-24C show the results of competitive binding experiments
examining anti-
MerTK antibodies. Anti-MerTK antibodies Y323, A3KCAT, 590H11G1E3 and MAB8912-
100 were
tested for competition with antibody h13B4.v16 for binding to human MerTK
using the classic
sandwich format (FIG. 24A). Antibody Y323 was found to compete with h13B4.v16
for binding to
human MerTK (FIG. 24B), whereas antibodies A3KCAT, 590H11G1E3 and MAB8912-100
did not
compete with h13B4.v16 for binding to human MerTK (FIG. 24C).
DETAILED DESCRIPTION
I. Definitions
[0084] It is to be understood that this disclosure is not limited to
particular compositions or
biological systems, which can, of course, vary. It is also to be understood
that the terminology used
herein is for the purpose of describing particular embodiments only, and is
not intended to be limiting.
[0085] As used in this specification and the appended claims, the singular
forms "a", "an" and
"the" include plural referents unless the content clearly dictates otherwise.
Thus, for example,
reference to "a molecule" optionally includes a combination of two or more
such molecules, and the
like.
[0086] The term "about" as used herein refers to the usual error range for
the respective value
readily known to the skilled person in this technical field. Reference to
"about" a value or parameter
herein includes (and describes) embodiments that are directed to that value or
parameter per se.
[0087] It is understood that aspects and embodiments of the present
disclosure include
comprising," "consisting," and "consisting essentially of' aspects and
embodiments.
[0088] An "acceptor human framework" for the purposes herein is a framework
comprising the
amino acid sequence of a light chain variable domain (VL) framework or a heavy
chain variable
domain (VH) framework derived from a human immunoglobulin framework or a human
consensus
framework, as defined below. An acceptor human framework "derived from" a
human
immunoglobulin framework or a human consensus framework may comprise the same
amino acid
sequence thereof, or it may contain amino acid sequence changes. In some
embodiments, the number
of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or
less, 5 or less, 4 or less, 3 or
less, or 2 or less, or 1 or less. In some embodiments, the VL acceptor human
framework is identical
in sequence to the VL human immunoglobulin framework sequence or human
consensus framework
sequence. In some embodiments, the VH acceptor human framework is identical in
sequence to the
VH human immunoglobulin framework sequence or human consensus framework
sequence. In some
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embodiments, the VL and VH acceptor human frameworks are identical in sequence
to a VH and VL
human immunoglobulin framework sequence or human consensus framework sequence.
[0089] "Affinity" refers to the strength of the sum total of noncovalent
interactions between a
single binding site of a molecule (e.g., an antibody) and its binding partner
(e.g., an antigen). Unless
indicated otherwise, as used herein, "binding affinity" refers to intrinsic
binding affinity which
reflects a 1:1 interaction between members of a binding pair (e.g., antibody
and antigen). The affinity
of a molecule X for its partner Y can generally be represented by the
dissociation constant (Kd).
Affinity can be measured by common methods known in the art, including those
described herein.
Specific illustrative and exemplary embodiments for measuring binding affinity
are described in the
following.
[0090] An "affinity matured" antibody refers to an antibody with one or
more alterations in one
or more hypervariable regions (HVRs), compared to a parent antibody which does
not possess such
alterations, such alterations resulting in an improvement in the affinity of
the antibody for antigen.
[0091] The terms "anti-MerTK antibody" and "an antibody that binds to
MerTK" refer to an
antibody that is capable of binding MerTK with sufficient affinity such that
the antibody is useful as a
diagnostic and/or therapeutic agent in targeting MerTK. In one embodiment, the
extent of binding of
an anti-MerTK antibody to an unrelated, non-MerTK protein is less than about
10% of the binding of
the antibody to MerTK as measured, e.g., by a radioimmunoassay (RIA). In
certain embodiments, an
antibody that binds to MerTK has a dissociation constant (Kd) of < 1 M, < 100
nM, < 10 nM, < 1
nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10' M or less, e.g. from 10' M to
10-13 M, e.g., from
tr9 tvt to 1 tr13 M). In certain embodiments, an anti-MerTK antibody binds to
an epitope of MerTK
that is conserved among MerTK from different species.
[0092] The term "antibody" herein is used in the broadest sense and
encompasses various
antibody structures, including but not limited to monoclonal antibodies,
polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and antibody fragments
so long as they exhibit
the desired antigen-binding activity.
[0093] An "antibody fragment" refers to a molecule other than an intact
antibody that comprises
a portion of an intact antibody that binds the antigen to which the intact
antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH,
F(ab')2; diabodies; linear
antibodies; single-chain antibody molecules (e.g. scFv); and multispecific
antibodies formed from
antibody fragments.
[0094] The term "chimeric" antibody refers to an antibody in which a
portion of the heavy and/or
light chain is derived from a particular source or species, while the
remainder of the heavy and/or
light chain is derived from a different source or species.
[0095] The "class" of an antibody refers to the type of constant domain or
constant region
possessed by its heavy chain. There are five major classes of antibodies: IgA,
IgD, IgE, IgG, and
IgM, and several of these may be further divided into subclasses (isotypes),
e.g., IgGl, IgG2, IgG3,
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IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the
different classes of
immunoglobulins are called cc, 6, c, y, and t, respectively.
[0096] The term "cytotoxic agent" as used herein refers to a substance that
inhibits or prevents a
cellular function and/or causes cell death or destruction. Cytotoxic agents
include, but are not limited
to, radioactive isotopes (e.g., At2",J131, I125, Y90, Re186, Re188, sm153,
Bi212, P32, p22
and radioactive
isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate,
adriamicin, vinca alkaloids
(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil,
daunorubicin or other intercalating agents); growth inhibitory agents; enzymes
and fragments thereof
such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins
or enzymatically active
toxins of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the
various antitumor or anticancer agents disclosed below.
[0097] "Effector functions" refer to those biological activities
attributable to the Fc region of an
antibody, which vary with the antibody isotype. Examples of antibody effector
functions include:
Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding;
antibody-
dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of
cell surface
receptors (e.g. B cell receptor); and B cell activation.
[0098] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an
amount effective, at dosages and for periods of time necessary, to achieve the
desired therapeutic or
prophylactic result.
[0099] The term "Fc region" herein is used to define a C-terminal region of
an immunoglobulin
heavy chain that contains at least a portion of the constant region. The term
includes native sequence
Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain
Fc region extends
from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless otherwise
specified herein,
numbering of amino acid residues in the Fc region or constant region is
according to the EU
numbering system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, MD,
1991.
[0100] "Framework" or "FR" refers to variable domain residues other than
hypervariable region
(HVR) residues. The FR of a variable domain generally consists of four FR
domains: FR1, FR2,
FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence
in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0101] The terms "full length antibody," "intact antibody," and "whole
antibody" are used herein
interchangeably to refer to an antibody having a structure substantially
similar to a native antibody
structure or having heavy chains that contain an Fc region as defined herein.
[0102] The terms "host cell," "host cell line," and "host cell culture" are
used interchangeably
and refer to cells into which exogenous nucleic acid has been introduced,
including the progeny of
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such cells. Host cells include "transformants" and "transformed cells," which
include the primary
transformed cell and progeny derived therefrom without regard to the number of
passages. Progeny
may not be completely identical in nucleic acid content to a parent cell, but
may contain mutations.
Mutant progeny that have the same function or biological activity as screened
or selected for in the
originally transformed cell are included herein.
[0103] A "human antibody" is one which possesses an amino acid sequence
which corresponds
to that of an antibody produced by a human or a human cell or derived from a
non-human source that
utilizes human antibody repertoires or other human antibody-encoding
sequences. This definition of a
human antibody specifically excludes a humanized antibody comprising non-human
antigen-binding
residues.
[0104] A "human consensus framework" is a framework which represents the
most commonly
occurring amino acid residues in a selection of human immunoglobulin VL or VH
framework
sequences. Generally, the selection of human immunoglobulin VL or VH sequences
is from a
subgroup of variable domain sequences. Generally, the subgroup of sequences is
a subgroup as in
Kabat etal., Sequences of Proteins of Immunological Interest, Fifth Edition,
NIH Publication 91-
3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for the VL, the
subgroup is subgroup
kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup
is subgroup III as in
Kabat et al., supra.
[0105] A "humanized" antibody refers to a chimeric antibody comprising
amino acid residues
from non-human HVRs and amino acid residues from human FRs. In certain
embodiments, a
humanized antibody will comprise substantially all of at least one, and
typically two, variable
domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond
to those of a non-
human antibody, and all or substantially all of the FRs correspond to those of
a human antibody. A
humanized antibody optionally may comprise at least a portion of an antibody
constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a non-human
antibody, refers to an
antibody that has undergone humanization.
[0106] The term "hypervariable region" or "HVR" as used herein refers to
each of the regions of
an antibody variable domain which are hypervariable in sequence
("complementarity determining
regions" or "CDRs") and/or form structurally defined loops ("hypervariable
loops") and/or contain
the antigen-contacting residues ("antigen contacts"). Generally, antibodies
comprise six HVRs: three
in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Exemplary HVRs
herein include:
(a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52
(L2), 91-96 (L3),
26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol.
196:901-917 (1987));
(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3),
31-35b (H1),
50-65 (H2), and 95-102 (H3) (Kabat etal., Sequences of Proteins of
Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, MD (1991));
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(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2),
89-96 (L3),
30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262:
732-745 (1996)); and
(d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-
56 (L2), 47-56
(L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102
(H3), and 94-102 (H3).
[0107] In one embodiment, HVR residues comprise those identified in TABLE 6
of the present
disclosure.
[0108] Unless otherwise indicated, HVR residues and other residues in the
variable domain (e.g.,
FR residues) are numbered herein according to Kabat et al., supra.
[0109] An "immunoconjugate" is an antibody conjugated to one or more
heterologous
molecule(s), including but not limited to a cytotoxic agent.
[0110] An "individual" or "subject" is a mammal. Mammals include, but are
not limited to,
domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates
(e.g., humans and non-
human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
In certain embodiments,
the individual or subject is a human.
[0111] An "isolated" antibody is one which has been separated from a
component of its natural
environment. In some embodiments, an antibody is purified to greater than 95%
or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric
focusing (IEF), capillary
electrophoresis) or chromatographic (e.g., ion exchange or reverse phase
HPLC). For review of
methods for assessment of antibody purity, see, e.g., Flatman etal., J.
Chromatogr. B 848:79-87
(2007).
[0112] An "isolated" nucleic acid refers to a nucleic acid molecule that
has been separated from a
component of its natural environment. An isolated nucleic acid includes a
nucleic acid molecule
contained in cells that ordinarily contain the nucleic acid molecule, but the
nucleic acid molecule is
present extrachromosomally or at a chromosomal location that is different from
its natural
chromosomal location.
[0113] "Isolated nucleic acid encoding an anti-MerTK antibody" refers to
one or more nucleic
acid molecules encoding antibody heavy and light chains (or fragments
thereof), including such
nucleic acid molecule(s) in a single vector or separate vectors, and such
nucleic acid molecule(s)
present at one or more locations in a host cell.
[0114] The term "LALAPG mutation" as used herein refers to a mutation in
the Fc region of an
antibody comprising the following three mutations: leucine 234 to alanine
(L234A), leucine 235 to
alanine (L235A), and proline 239 to glycine (P329G), which has previously been
shown to reduce
binding to Fc receptors and complement (see e.g., US Publication No.
2012/0251531 and US Patent
No. 8,969,526). The numbering of amino acid residues in the Fc region or
constant region is
according to the EU numbering system, also called the EU index, as described
in Kabat et al.,
Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service,
National Institutes of
Health, Bethesda, MD, 1991.
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[0115] The term "monoclonal antibody" as used herein refers to an antibody
obtained from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the
population are identical and/or bind the same epitope, except for possible
variant antibodies, e.g.,
containing naturally occurring mutations or arising during production of a
monoclonal antibody
preparation, such variants generally being present in minor amounts. In
contrast to polyclonal
antibody preparations, which typically include different antibodies directed
against different
determinants (epitopes), each monoclonal antibody of a monoclonal antibody
preparation is directed
against a single determinant on an antigen. Thus, the modifier "monoclonal"
indicates the character
of the antibody as being obtained from a substantially homogeneous population
of antibodies, and is
not to be construed as requiring production of the antibody by any particular
method. For example,
the monoclonal antibodies to be used in accordance with the present invention
may be made by a
variety of techniques, including but not limited to the hybridoma method,
recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals containing all
or part of the human
immunoglobulin loci, such methods and other exemplary methods for making
monoclonal antibodies
being described herein.
[0116] A "naked antibody" refers to an antibody that is not conjugated to a
heterologous moiety
(e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in
a pharmaceutical
formulation.
[0117] "Native antibodies" refer to naturally occurring immunoglobulin
molecules with varying
structures. For example, native IgG antibodies are heterotetrameric
glycoproteins of about 150,000
daltons, composed of two identical light chains and two identical heavy chains
that are disulfide-
bonded. From N- to C-terminus, each heavy chain has a variable region (VH),
also called a variable
heavy domain or a heavy chain variable domain, followed by three constant
domains (CHL CH2, and
CH3). Similarly, from N- to C-terminus, each light chain has a variable region
(VL), also called a
variable light domain or a light chain variable domain, followed by a constant
light (CL) domain. The
light chain of an antibody may be assigned to one of two types, called kappa
(K) and lambda (i),
based on the amino acid sequence of its constant domain.
[0118] The term "package insert" is used to refer to instructions
customarily included in
commercial packages of therapeutic products, that contain information about
the indications, usage,
dosage, administration, combination therapy, contraindications and/or warnings
concerning the use of
such therapeutic products.
[0119] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide
sequence is defined as the percentage of amino acid residues in a candidate
sequence that are identical
with the amino acid residues in the reference polypeptide sequence, after
aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent sequence
identity, and not
considering any conservative substitutions as part of the sequence identity.
Alignment for purposes of
determining percent amino acid sequence identity can be achieved in various
ways that are within the
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skill in the art, for instance, using publicly available computer software
such as BLAST, BLAST-2,
ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate
parameters for aligning sequences, including any algorithms needed to achieve
maximal alignment
over the full length of the sequences being compared. For purposes herein,
however, % amino acid
sequence identity values are generated using the sequence comparison computer
program ALIGN-2.
The ALIGN-2 sequence comparison computer program was authored by Genentech,
Inc., and the
source code has been filed with user documentation in the U.S. Copyright
Office, Washington D.C.,
20559, where it is registered under U.S. Copyright Registration No. TXU510087.
The ALIGN-2
program is publicly available from Genentech, Inc., South San Francisco,
California, or may be
compiled from the source code. The ALIGN-2 program should be compiled for use
on a UNIX
operating system, including digital UNIX V4.0D. All sequence comparison
parameters are set by the
ALIGN-2 program and do not vary.
[0120] In situations where ALIGN-2 is employed for amino acid sequence
comparisons, the %
amino acid sequence identity of a given amino acid sequence A to, with, or
against a given amino acid
sequence B (which can alternatively be phrased as a given amino acid sequence
A that has or
comprises a certain % amino acid sequence identity to, with, or against a
given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment
program ALIGN-2 in that program's alignment of A and B, and where Y is the
total number of amino
acid residues in B. It will be appreciated that where the length of amino acid
sequence A is not equal
to the length of amino acid sequence B, the % amino acid sequence identity of
A to B will not equal
the % amino acid sequence identity of B to A. Unless specifically stated
otherwise, all % amino acid
sequence identity values used herein are obtained as described in the
immediately preceding
paragraph using the ALIGN-2 computer program.
[0121] The term "PD-1 axis binding antagonist" refers to a molecule that
inhibits the interaction
of a PD-1 axis binding partner with either one or more of its binding
partners, so as to remove T-cell
dysfunction resulting from signaling on the PD-1 signaling axis ¨ with a
result being to restore or
enhance T-cell function (e.g., proliferation, cytokine production, target cell
killing). As used herein, a
PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-Li
binding antagonist and a
PD-L2 binding antagonist.
[0122] The term "PD-1 binding antagonist" refers to a molecule that
decreases, blocks, inhibits,
abrogates or interferes with signal transduction resulting from the
interaction of PD-1 with one or
more of its binding partners, such as PD-Li and/or PD-L2. In some embodiments,
the PD-1 binding
antagonist is a molecule that inhibits the binding of PD-1 to one or more of
its binding partners. In a
specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to
PD-Li and/or PD-L2. For
example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen
binding fragments thereof,
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immunoadhesins, fusion proteins, oligopeptides and other molecules that
decrease, block, inhibit,
abrogate or interfere with signal transduction resulting from the interaction
of PD-1 with PD-Li
and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces the
negative co-stimulatory
signal mediated by or through cell surface proteins expressed on T lymphocytes
that mediate
signaling through PD-1 so as to render a dysfunctional T-cell less
dysfunctional (e.g., enhancing
effector responses to antigen recognition). In some embodiments, the PD-1
binding antagonist is an
anti-PD-1 antibody. Specific examples of PD-1 binding antagonists are provided
infra.
[0123] The term "PD-Li binding antagonist" refers to a molecule that
decreases, blocks, inhibits,
abrogates or interferes with signal transduction resulting from the
interaction of PD-Li with either
one or more of its binding partners, such as PD-1 and/or B7-1. In some
embodiments, a PD-Li
binding antagonist is a molecule that inhibits the binding of PD-Li to its
binding partners. In a
specific aspect, the PD-Li binding antagonist inhibits binding of PD-Li to PD-
1 and/or B7-1. In
some embodiments, the PD-Li binding antagonists include anti-PD-Li antibodies,
antigen binding
fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other
molecules that decrease,
block, inhibit, abrogate or interfere with signal transduction resulting from
the interaction of PD-Li
with one or more of its binding partners, such as PD-1 and/or B7-1. In one
embodiment, a PD-Li
binding antagonist reduces the negative co-stimulatory signal mediated by or
through cell surface
proteins expressed on T lymphocytes that mediate signaling through PD-Li so as
to render a
dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to
antigen recognition). In
some embodiments, a PD-Li binding antagonist is an anti-PD-Li antibody.
Specific examples of PD-
Li binding antagonists are provided infra.
[0124] The term "PD-L2 binding antagonist" refers to a molecule that
decreases, blocks, inhibits,
abrogates or interferes with signal transduction resulting from the
interaction of PD-L2 with either
one or more of its binding partners, such as PD-1. In some embodiments, a PD-
L2 binding antagonist
is a molecule that inhibits the binding of PD-L2 to one or more of its binding
partners. In a specific
aspect, the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1. In
some embodiments, the
PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments
thereof,
immunoadhesins, fusion proteins, oligopeptides and other molecules that
decrease, block, inhibit,
abrogate or interfere with signal transduction resulting from the interaction
of PD-L2 with one or
more of its binding partners, such as PD-1. In one embodiment, a PD-L2 binding
antagonist reduces
the negative co-stimulatory signal mediated by or through cell surface
proteins expressed on T
lymphocytes that mediate signaling through PD-L2 so as to render a
dysfunctional T-cell less
dysfunctional (e.g., enhancing effector responses to antigen recognition). In
some embodiments, a
PD-L2 binding antagonist is an immunoadhesin.
[0125] The term "pharmaceutical formulation" refers to a preparation which
is in such form as to
permit the biological activity of an active ingredient contained therein to be
effective, and which
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contains no additional components which are unacceptably toxic to a subject to
which the formulation
would be administered.
[0126] A "pharmaceutically acceptable carrier" refers to an ingredient in a
pharmaceutical
formulation, other than an active ingredient, which is nontoxic to a subject.,
A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer, excipient,
stabilizer, or preservative.
[0127] The term "MerTK," as used herein, refers to any native MerTK from
any vertebrate
source, including mammals such as primates (e.g. humans) and rodents (e.g.,
mice and rats), unless
otherwise indicated. The term encompasses "full-length," unprocessed MerTK as
well as any form of
MerTK that results from processing in the cell. The term also encompasses
naturally occurring
variants of MerTK, e.g., splice variants or allelic variants. The amino acid
sequence of an exemplary
human MerTK is described in US 2006/0121562.
[0128] As used herein, "treatment" (and grammatical variations thereof such
as "treat" or
"treating") refers to clinical intervention in an attempt to alter the natural
course of the individual
being treated, and can be performed either for prophylaxis or during the
course of clinical pathology.
Desirable effects of treatment include, but are not limited to, preventing
occurrence or recurrence of
disease, alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or
palliation of the disease state, and remission or improved prognosis. In some
embodiments,
antibodies of the invention are used to delay development of a disease or to
slow the progression of a
disease.
[0129] The term "variable region" or "variable domain" refers to the domain
of an antibody
heavy or light chain that is involved in binding of the antibody to antigen.
The variable domains of
the heavy chain and light chain (VH and VL, respectively) of a native antibody
generally have similar
structures, with each domain comprising four conserved framework regions (FRs)
and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th
ed., W.H. Freeman and
Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer
antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen may be
isolated using a VH or VL
domain from an antibody that binds the antigen to screen a library of
complementary VL or VH
domains, respectively. See, e.g., Portolano etal., J. Immunol. 150:880-887
(1993); Clarkson etal.,
Nature 352:624-628 (1991).
[0130] The term "vector," as used herein, refers to a nucleic acid molecule
capable of
propagating another nucleic acid to which it is linked. The term includes the
vector as a self-
replicating nucleic acid structure as well as the vector incorporated into the
genome of a host cell into
which it has been introduced. Certain vectors are capable of directing the
expression of nucleic acids
to which they are operatively linked. Such vectors are referred to herein as
"expression vectors."
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II. Anti-MerTK Antibodies
[0131] The present disclosure is based on the discovery of novel anti-MerTK
antibodies. Such
novel anti-MerTK antibodies find use in the treatment of cancer. In
particular, the present disclosure
is based on the discovery that the anti-MerTK antibodies described herein
enhance the effectiveness
of immune checkpoint inhibitor-based therapy.
[0132] C-Mer proto-oncogene tyrosine kinase (MerTK) is a receptor tyrosine
kinase which
transduces extracellular signals upon binding to various ligands, such as
galectin-3, Protein S, and
Gas6, thus activating expression of effector genes. The MerTK pathway
regulates essential cellular
processes, including cell survival, cytokine production, migration,
differentiation, and phagocytosis
(Cabernoy N., etal. J Cell Physio. 227 (2012): 401-407; Wu, G., etal. Cell
Death & Disease 8
(2017): e2700). Expression of MerTK is found in a variety of hematopoeietic
cell types, such as
macrophages, dendritic cells, natural killer (NK) cells. Importantly, the
MerTK receptor pathway is
active in several solid and hematological cancers, including colon cancer (Wu,
G., etal. Cell Death &
Disease 8 (2017): e2700).
[0133] The MerTK receptor is composed of an extracellular component, a
transmembrane (TM)
domain, and an intracellular component. As shown in the diagram below, the
extracellular or ligand-
binding region of MerTK contains two immunoglobulin (Ig)-like domains and two
fibronectin (FN)
type III-like domains.
1g-like FN-like
Domain Domain Doniaai Domaiti Dcrtmitl TK Domain
NET2 COOki
[0134] In human MerTK, for example, the two Ig-like domains are defined by
amino acid
residues 76-195 and amino acid residues 199-283, respectively. Additionally,
the two fibronectin-like
domains of human MerTK are defined by amino acid residues 286-384 and amino
acid residues 388-
480, respectively. The intracellular region of MerTK contains a tyrosine
kinase (TK) domain, which
autophosphorylates specific tyrosine residues following ligand binding to the
extracellular region and
facilitates MerTK receptor dimerization, thus activating downstream effector
gene expression
(Toledo, R.A, etal. Clin Can. Res. 22 (2016): 2301-2312). Human MerTK
comprises the amino acid
sequence:
MGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPAL
MFSPTQPGRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIIL SEHKGVKFNCSISVPNIYQDTTI
SWWKDGKELLGAHHAITQFYPDDEVTAHASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEV
QGLPHFTKQPESMNVTRNTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTE
MAVFSCEAHNDKGLTVSKGVQINIKAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQV
KEADPLSNGSVMIFNTSALPHLYQIKQLQALANYSIGVSCMNEIGWSAVSPWILASTTEGAPS
VAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGYRISHVWQSAGISKELLEEVGQNGSR
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ARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAP SSTPAPGNADPVLIIFGCF
CGFILIGLILYISLAIRKRVQETKFGNAFTEED SELVVNYIAKKSFCRRAIELTLHSLGVSEELQN
KLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGT SLKVAVKTMKLDNSSQREIEEFLSEA
ACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTLL
KFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGL SKKIYSGDYYRQGRIAK
MPVKWIAIESLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQ
PEDCLDELYEIMYSCWRTDPLDRPTFSVLRLQLEKLLESLPDVRNQADVIYVNTQLLESSEGL
AQGSTLAPLDLNIDPDSIIASCTPRAAISVVTAEVHDSKPHEGRYILNGGSEEWEDLT SAP SAA
VTAEKNSVLPGERLVRNGVSWSHSSMLPLGSSLPDELLFADDSSEGSEVLM (SEQ ID NO:
137).
[0135] Provided herein are isolated antibodies that bind to MerTK, wherein
the antibodies have
one or more of the following properties: (i) antagonizes one or more
biological activities of MerTK,
(ii) reduces MerTK-mediated clearance of apoptotic cells, (iii) reduces MerTK-
mediated phagocytic
activity, (iv) enhances tumor immunogenicity of a checkpoint inhibitor, (v)
binds to a fibronectin-like
domain of MerTK, (vi) binds to an Ig-like domain on MerTK, (vii) binds
specifically to human
MerTK, (viii) binds to one or more of human, mouse and/or cyno MerTK, and/or
(ix) binds to MerTK
with a KID of less than 20 nM (e.g., less than 10 nM, less than 5 nM, or less
than 2 nM).
A. Exemplary Anti-MerTK Antibodies
[0136] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising
the amino acid
sequence of SEQ ID NO: 4; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO: 5; and
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In one
embodiment, the
antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:
4; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO: 5; and (c) HVR-H3 comprising
the amino acid
sequence of SEQ ID NO: 6. In an exemplary embodiment, the anti-MerTK antibody
binds to a
fibronectin-like domain of MerTK.
[0137] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-L1
comprising the amino acid
sequence of SEQ ID NO: 1; (b) HVR-L2 comprising the amino acid sequence of SEQ
ID NO: 2; and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 3. In one
embodiment, the antibody
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 1; (b)
HVR-L2
comprising the amino acid sequence of SEQ ID NO: 2; and (c) HVR-L3 comprising
the amino acid
sequence of SEQ ID NO: 3. In an exemplary embodiment, the anti-MerTK antibody
binds to a
fibronectin-like domain of MerTK.
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[0138] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 4, (ii) HVR-H2 comprising the
amino acid
sequence of SEQ ID NO: 5, and (iii) HVR-H3 comprising an amino acid sequence
selected from SEQ
ID NO: 6; and (b) a VL domain comprising at least one, at least two, or all
three VL HVR sequences
selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 1,
(ii) HVR-L2
comprising the amino acid sequence of SEQ ID NO: 2, and (c) HVR-L3 comprising
the amino acid
sequence of SEQ ID NO: 3. In an exemplary embodiment, the anti-MerTK antibody
binds to a
fibronectin-like domain of MerTK.
[0139] In another aspect, the invention provides an antibody comprising (a)
HVR-H1 comprising
the amino acid sequence of SEQ ID NO: 4; (b) HVR-H2 comprising the amino acid
sequence of SEQ
ID NO: 5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d)
HVR-L1
comprising the amino acid sequence of SEQ ID NO: 1; (e) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 2; and (f) HVR-L3 comprising an amino acid sequence
selected from SEQ
ID NO: 3. In an exemplary embodiment, the anti-MerTK antibody binds to a
fibronectin-like domain
of MerTK.
[0140] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins oflintnunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0141] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 83. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 83. In certain embodiments, substitutions, insertions, or
deletions occur in regions
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outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 83, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 4, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 5, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
[0142] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 65. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 65. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 65, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-Li
comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 2; and (c) HVR-L3 comprising the amino acid sequence of
SEQ ID NO: 3.
In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of MerTK.
[0143] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 83 and
SEQ ID NO: 65, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0144] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-Hl comprising
the amino acid
sequence of SEQ ID NO: 10; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 11;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12. In one
embodiment, the
antibody comprises (a) HVR-Hl comprising the amino acid sequence of SEQ ID NO:
10; (b) HVR-
H2 comprising the amino acid sequence of SEQ ID NO: 11; and (c) HVR-H3
comprising the amino
acid sequence of SEQ ID NO: 12. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0145] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-Li
comprising the amino acid
sequence of SEQ ID NO:7; (b) HVR-L2 comprising the amino acid sequence of SEQ
ID NO: 8; and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 9. In one
embodiment, the antibody
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comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (b)
HVR-L2
comprising the amino acid sequence of SEQ ID NO: 8; and (c) HVR-L3 comprising
the amino acid
sequence of SEQ ID NO: 9. In an exemplary embodiment, the anti-MerTK antibody
binds to a
fibronectin-like domain of MerTK.
[0146] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 10, (ii) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 11, and (iii) HVR-H3 comprising an amino acid sequence
selected from
SEQ ID NO: 12; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 7, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 9. In an exemplary embodiment, the anti-
MerTK antibody
binds to a fibronectin-like domain of MerTK.
[0147] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 10; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 11; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 12;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 8; and (f) HVR-L3 comprising an amino acid
sequence selected
from SEQ ID NO: 9. In an exemplary embodiment, the anti-MerTK antibody binds
to a fibronectin-
like domain of MerTK.
[0148] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins ofinnnunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0149] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 84. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
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substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 84. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 84, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 10, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 11, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
[0150] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 66. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 66, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 8; and (c) HVR-L3 comprising the amino acid sequence of
SEQ ID NO: 9.
In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of MerTK.
[0151] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 84 and
SEQ ID NO: 66, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0152] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 85. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 85. In certain embodiments, substitutions, insertions, or
deletions occur in regions
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outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 85, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 10, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 11, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
[0153] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 67. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 67. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 67, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 8; and (c) HVR-L3 comprising the amino acid sequence of
SEQ ID NO: 9.
[0154] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 85 and
SEQ ID NO: 67, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0155] In another aspect, an anti-MerTK antibody comprises a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 102. In certain embodiments, a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to the
reference sequence, but an anti-
MerTK antibody comprising that sequence retains the ability to bind to MerTK.
In certain
embodiments, a total of 1 to 10 amino acids have been substituted, inserted
and/or deleted in SEQ ID
NO: 102. In certain embodiments, substitutions, insertions, or deletions occur
in regions outside the
HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody comprises the
heavy chain sequence in
SEQ ID NO: 102, including post-translational modifications of that sequence.
In a particular
embodiment, the heavy chain comprises one, two or three HVRs selected from:
(a) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 10, (b) HVR-H2 comprising the
amino acid
sequence of SEQ ID NO: 11, and (c) HVR-H3 comprising the amino acid sequence
of SEQ ID NO:
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12. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0156] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 110. In certain
embodiments, a light
chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity
contains substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the
reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to bind
to MerTK. In certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted
and/or deleted in SEQ ID NO: 110. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the light chain sequence in SEQ ID NO: 110, including post-translational
modifications of that
sequence. In a particular embodiment, the light chain comprises one, two or
three HVRs selected
from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 8; and (c) HVR-L3 comprising the amino acid
sequence of SEQ
ID NO: 9. In an exemplary embodiment, the anti-MerTK antibody binds to a
fibronectin-like domain
of MerTK.
[0157] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
heavy chain as in any of the embodiments provided above, and a light chain as
in any of the
embodiments provided above. In one embodiment, the antibody comprises the
heavy chain and light
chain sequences in SEQ ID NO: 102 and SEQ ID NO: 110, respectively, including
post-translational
modifications of those sequences. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0158] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 86. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 86. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 86, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 10, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 11, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 12. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
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[0159] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 68. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 68. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 68, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 8; and (c) HVR-L3 comprising the amino acid sequence of
SEQ ID NO: 9.
In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of MerTK.
[0160] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 86 and
SEQ ID NO: 68, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0161] In another aspect, an anti-MerTK antibody comprises a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 103. In certain embodiments, a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to the
reference sequence, but an anti-
MerTK antibody comprising that sequence retains the ability to bind to MerTK.
In certain
embodiments, a total of 1 to 10 amino acids have been substituted, inserted
and/or deleted in SEQ ID
NO: 103. In certain embodiments, substitutions, insertions, or deletions occur
in regions outside the
HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody comprises the
heavy chain sequence in
SEQ ID NO: 103, including post-translational modifications of that sequence.
In a particular
embodiment, the heavy chain comprises one, two or three HVRs selected from:
(a) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 10, (b) HVR-H2 comprising the
amino acid
sequence of SEQ ID NO: 11, and (c) HVR-H3 comprising the amino acid sequence
of SEQ ID NO:
12. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0162] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 111. In certain
embodiments, a light
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chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity
contains substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the
reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to bind
to MerTK. In certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted
and/or deleted in SEQ ID NO: 111. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the light chain sequence in SEQ ID NO: 111, including post-translational
modifications of that
sequence. In a particular embodiment, the light chain comprises one, two or
three HVRs selected
from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 8; and (c) HVR-L3 comprising the amino acid
sequence of SEQ
ID NO: 9. In an exemplary embodiment, the anti-MerTK antibody binds to a
fibronectin-like domain
of MerTK.
[0163] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
heavy chain as in any of the embodiments provided above, and a light chain as
in any of the
embodiments provided above. In one embodiment, the antibody comprises the
heavy chain and light
chain sequences in SEQ ID NO: 103 and SEQ ID NO: 111, respectively, including
post-translational
modifications of those sequences. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0164] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising
the amino acid
sequence of SEQ ID NO: 16; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 17;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18. In one
embodiment, the
antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:
16; (b) HVR-
H2 comprising the amino acid sequence of SEQ ID NO: 17; and (c) HVR-H3
comprising the amino
acid sequence of SEQ ID NO: 18. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0165] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-L1
comprising the amino acid
sequence of SEQ ID NO: 13; (b) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 14;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 15. In one
embodiment, the
antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:
13; (b) HVR-
L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3
comprising the amino
acid sequence of SEQ ID NO: 15. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0166] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 16, (ii) HVR-H2 comprising
the amino acid
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sequence of SEQ ID NO: 17, and (iii) HVR-H3 comprising an amino acid sequence
selected from
SEQ ID NO: 18; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 13, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 15. In an exemplary embodiment, the anti-
MerTK antibody
binds to a fibronectin-like domain of MerTK.
[0167] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 16; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 17; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 18;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising an amino acid
sequence
selected from SEQ ID NO: 15. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0168] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins ofinnnunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0169] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 87. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 87. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 87, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hl
comprising the
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amino acid sequence of SEQ ID NO: 16, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 17, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
[0170] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 69. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 69. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 69, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
15. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0171] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 87 and
SEQ ID NO: 69, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0172] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 88. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 88. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 88, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hl
comprising the
amino acid sequence of SEQ ID NO: 16, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 17, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
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101731 In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 70. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 70. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 70, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
15. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
101741 In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 88 and
SEQ ID NO: 70, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
101751 In another aspect, an anti-MerTK antibody comprises a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 104. In certain embodiments, a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to the
reference sequence, but an anti-
MerTK antibody comprising that sequence retains the ability to bind to MerTK.
In certain
embodiments, a total of 1 to 10 amino acids have been substituted, inserted
and/or deleted in SEQ ID
NO: 104. In certain embodiments, substitutions, insertions, or deletions occur
in regions outside the
HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody comprises the
heavy chain sequence in
SEQ ID NO: 104, including post-translational modifications of that sequence.
In a particular
embodiment, the heavy chain comprises one, two or three HVRs selected from:
(a) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 16, (b) HVR-H2 comprising the
amino acid
sequence of SEQ ID NO: 17, and (c) HVR-H3 comprising the amino acid sequence
of SEQ ID NO:
18. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
101761 In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%
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sequence identity to the amino acid sequence of SEQ ID NO: 112. In certain
embodiments, a light
chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity
contains substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the
reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to bind
to MerTK. In certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted
and/or deleted in SEQ ID NO: 112. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the light chain sequence in SEQ ID NO: 112, including post-translational
modifications of that
sequence. In a particular embodiment, the light chain comprises one, two or
three HVRs selected
from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-
L2 comprising
the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino
acid sequence of
SEQ ID NO: 15. In an exemplary embodiment, the anti-MerTK antibody binds to a
fibronectin-like
domain of MerTK.
101771 In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
heavy chain as in any of the embodiments provided above, and a light chain as
in any of the
embodiments provided above. In one embodiment, the antibody comprises the
heavy chain and light
chain sequences in SEQ ID NO: 104 and SEQ ID NO: 112, respectively, including
post-translational
modifications of those sequences. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0178] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 89. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 89. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 89, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 16, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 17, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
101791 In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 70. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
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99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 70. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 70, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
15. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0180] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 89 and
SEQ ID NO: 70, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0181] In another aspect, an anti-MerTK antibody comprises a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 105. In certain embodiments, a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to the
reference sequence, but an anti-
MerTK antibody comprising that sequence retains the ability to bind to MerTK.
In certain
embodiments, a total of 1 to 10 amino acids have been substituted, inserted
and/or deleted in SEQ ID
NO: 105. In certain embodiments, substitutions, insertions, or deletions occur
in regions outside the
HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody comprises the
heavy chain sequence in
SEQ ID NO: 105, including post-translational modifications of that sequence.
In a particular
embodiment, the heavy chain comprises one, two or three HVRs selected from:
(a) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 16, (b) HVR-H2 comprising the
amino acid
sequence of SEQ ID NO: 17, and (c) HVR-H3 comprising the amino acid sequence
of SEQ ID NO:
18. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0182] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 113. In certain
embodiments, a light
chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity
contains substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the
reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to bind
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to MerTK. In certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted
and/or deleted in SEQ ID NO: 113. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the light chain sequence in SEQ ID NO: 113, including post-translational
modifications of that
sequence. In a particular embodiment, the light chain comprises one, two or
three HVRs selected
from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 13; (b) HVR-
L2 comprising
the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino
acid sequence of
SEQ ID NO: 15. In an exemplary embodiment, the anti-MerTK antibody binds to a
fibronectin-like
domain of MerTK.
[0183] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
heavy chain as in any of the embodiments provided above, and a light chain as
in any of the
embodiments provided above. In one embodiment, the antibody comprises the
heavy chain and light
chain sequences in SEQ ID NO: 105 and SEQ ID NO: 113, respectively, including
post-translational
modifications of those sequences. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0184] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising
the amino acid
sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 23;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In one
embodiment, the
antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:
22; (b) HVR-
H2 comprising the amino acid sequence of SEQ ID NO: 23; and (c) HVR-H3
comprising the amino
acid sequence of SEQ ID NO: 24. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0185] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-L1
comprising the amino acid
sequence of SEQ ID NO: 19; (b) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 20;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In one
embodiment, the
antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:
19; (b) HVR-
L2 comprising the amino acid sequence of SEQ ID NO: 20; and (c) HVR-L3
comprising the amino
acid sequence of SEQ ID NO: 21. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0186] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 22, (ii) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 23, and (iii) HVR-H3 comprising an amino acid sequence
selected from
SEQ ID NO: 24; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 19, (ii)
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HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 21. In an exemplary embodiment, the anti-
MerTK antibody
binds to a fibronectin-like domain of MerTK.
[0187] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 23; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 24;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 20; and (f) HVR-L3 comprising an amino acid
sequence
selected from SEQ ID NO: 21. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0188] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0189] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 90. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 90. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 90, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hl
comprising the
amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 23, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
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[0190] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 71. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 71, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 19; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
21. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0191] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 90 and
SEQ ID NO: 71, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0192] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 91. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 91. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 91, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hl
comprising the
amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 23, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
[0193] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72. In
certain
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embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 72. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 72, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 19; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
21. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0194] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 91 and
SEQ ID NO: 72, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0195] In another aspect, an anti-MerTK antibody comprises a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 106. In certain embodiments, a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to the
reference sequence, but an anti-
MerTK antibody comprising that sequence retains the ability to bind to MerTK.
In certain
embodiments, a total of 1 to 10 amino acids have been substituted, inserted
and/or deleted in SEQ ID
NO: 106. In certain embodiments, substitutions, insertions, or deletions occur
in regions outside the
HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody comprises the
heavy chain sequence in
SEQ ID NO: 106, including post-translational modifications of that sequence.
In a particular
embodiment, the heavy chain comprises one, two or three HVRs selected from:
(a) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 comprising the
amino acid
sequence of SEQ ID NO: 23, and (c) HVR-H3 comprising the amino acid sequence
of SEQ ID NO:
24. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0196] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 114. In certain
embodiments, a light
chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity
contains substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the
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reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to bind
to MerTK. In certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted
and/or deleted in SEQ ID NO: 114. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the light chain sequence in SEQ ID NO: 114, including post-translational
modifications of that
sequence. In a particular embodiment, the light chain comprises one, two or
three HVRs selected
from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19; (b) HVR-
L2 comprising
the amino acid sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino
acid sequence of
SEQ ID NO: 21. In an exemplary embodiment, the anti-MerTK antibody binds to a
fibronectin-like
domain of MerTK.
[0197] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
heavy chain as in any of the embodiments provided above, and a light chain as
in any of the
embodiments provided above. In one embodiment, the antibody comprises the
heavy chain and light
chain sequences in SEQ ID NO: 106 and SEQ ID NO: 114, respectively, including
post-translational
modifications of those sequences. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0198] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 92. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 92. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 92, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 23, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
[0199] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 73. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
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and/or deleted in SEQ ID NO: 73. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 73, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1
comprising the amino acid sequence of SEQ ID NO: 19; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
21. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0200] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 92 and
SEQ ID NO: 73, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0201] In another aspect, an anti-MerTK antibody comprises a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 107. In certain embodiments, a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to the
reference sequence, but an anti-
MerTK antibody comprising that sequence retains the ability to bind to MerTK.
In certain
embodiments, a total of 1 to 10 amino acids have been substituted, inserted
and/or deleted in SEQ ID
NO: 107. In certain embodiments, substitutions, insertions, or deletions occur
in regions outside the
HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody comprises the
heavy chain sequence in
SEQ ID NO: 107, including post-translational modifications of that sequence.
In a particular
embodiment, the heavy chain comprises one, two or three HVRs selected from:
(a) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 22, (b) HVR-H2 comprising the
amino acid
sequence of SEQ ID NO: 23, and (c) HVR-H3 comprising the amino acid sequence
of SEQ ID
NO:24. In an exemplary embodiment, the anti-MerTK antibody binds to a
fibronectin-like domain of
MerTK.
[0202] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 115. In certain
embodiments, a light
chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity
contains substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the
reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to bind
to MerTK. In certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted
and/or deleted in SEQ ID NO: 115. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
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the light chain sequence in SEQ ID NO: 115, including post-translational
modifications of that
sequence. In a particular embodiment, the light chain comprises one, two or
three HVRs selected
from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19; (b) HVR-
L2 comprising
the amino acid sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino
acid sequence of
SEQ ID NO: 20. In an exemplary embodiment, the anti-MerTK antibody binds to a
fibronectin-like
domain of MerTK.
[0203] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
heavy chain as in any of the embodiments provided above, and a light chain as
in any of the
embodiments provided above. In one embodiment, the antibody comprises the
heavy chain and light
chain sequences in SEQ ID NO: 107 and SEQ ID NO: 115, respectively, including
post-translational
modifications of those sequences. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0204] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising
the amino acid
sequence of SEQ ID NO: 27; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 28;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29. In one
embodiment, the
antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:
27; (b) HVR-
H2 comprising the amino acid sequence of SEQ ID NO: 28; and (c) HVR-H3
comprising the amino
acid sequence of SEQ ID NO: 29. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0205] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-L1
comprising the amino acid
sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 14;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In one
embodiment, the
antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:
25; (b) HVR-
L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3
comprising the amino
acid sequence of SEQ ID NO: 26. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0206] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 27, (ii) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 28, and (iii) HVR-H3 comprising an amino acid sequence
selected from
SEQ ID NO: 29; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 25, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 26. In an exemplary embodiment, the anti-
MerTK antibody
binds to a fibronectin-like domain of MerTK.
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[0207] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 27; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 28; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 29;
(d) HVR-L 1 comprising the amino acid sequence of SEQ ID NO: 25; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising an amino acid
sequence
selected from SEQ ID NO: 26. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0208] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins ofinnnunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0209] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 93. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 93. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 93, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hl
comprising the
amino acid sequence of SEQ ID NO: 27, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 28, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
[0210] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74. In
certain
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embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 74. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 74, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-Li
comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
26. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0211] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 93 and
SEQ ID NO:74, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0212] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-Hl comprising
the amino acid
sequence of SEQ ID NO:33; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:34;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:35. In one
embodiment, the
antibody comprises (a) HVR-Hl comprising the amino acid sequence of SEQ ID
NO:33; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:34; and (c) HVR-H3 comprising
the amino acid
sequence of SEQ ID NO:35. In an exemplary embodiment, the anti-MerTK antibody
binds to a
fibronectin-like domain of MerTK.
[0213] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-Li
comprising the amino acid
sequence of SEQ ID NO: 30; (b) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 31;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 32. In one
embodiment, the
antibody comprises (a) HVR-Li comprising the amino acid sequence of SEQ ID NO:
30; (b) HVR-
L2 comprising the amino acid sequence of SEQ ID NO: 31; and (c) HVR-L3
comprising the amino
acid sequence of SEQ ID NO: 32. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0214] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 33, (ii) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 34, and (iii) HVR-H3 comprising an amino acid sequence
selected from
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SEQ ID NO: 35; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 30, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 32. In an exemplary embodiment, the anti-
MerTK antibody
binds to a fibronectin-like domain of MerTK.
[0215] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 34; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 35;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 30; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 31; and (f) HVR-L3 comprising an amino acid
sequence
selected from SEQ ID NO: 32. In an exemplary embodiment, the anti-MerTK
antibody binds to a
fibronectin-like domain of MerTK.
[0216] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins ofinnnunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0217] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 94. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 94. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 94, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hl
comprising the
amino acid sequence of SEQ ID NO: 33, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
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NO: 34, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 35. In
an exemplary
embodiment, the anti-MerTK antibody binds to a fibronectin-like domain of
MerTK.
[0218] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 75. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 75. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 75, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-Li
comprising the amino acid sequence of SEQ ID NO: 30; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 31; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
32. In an exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-
like domain of
MerTK.
[0219] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 94 and
SEQ ID NO: 75, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a fibronectin-like
domain of MerTK.
[0220] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-Hl comprising
the amino acid
sequence of SEQ ID NO: 38; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 39;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40. In one
embodiment, the
antibody comprises (a) HVR-Hl comprising the amino acid sequence of SEQ ID NO:
38; (b) HVR-
H2 comprising the amino acid sequence of SEQ ID NO: 39; and (c) HVR-H3
comprising the amino
acid sequence of SEQ ID NO: 40. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0221] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-Li
comprising the amino acid
sequence of SEQ ID NO: 36; (b) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 14;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 37. In one
embodiment, the
antibody comprises (a) HVR-Li comprising the amino acid sequence of SEQ ID NO:
36; (b) HVR-
L2 comprising the amino acid sequence of SEQ ID NO: 14; and (c) HVR-L3
comprising the amino
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acid sequence of SEQ ID NO: 37. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0222] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 38, (ii) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 39, and (iii) HVR-H3 comprising an amino acid sequence
selected from
SEQ ID NO: 40; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 36, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 37. In an exemplary embodiment, the anti-
MerTK antibody
binds to a Ig-like domain of MerTK.
[0223] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 39; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 40;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 36; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 14; and (f) HVR-L3 comprising an amino acid
sequence
selected from SEQ ID NO: 37. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0224] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins ofinnnunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0225] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 95. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
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In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 95. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 95, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 38, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 39, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40. In
an exemplary
embodiment, the anti-MerTK antibody binds to a Ig-like domain of MerTK.
[0226] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 76. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 76. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 76, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-Li
comprising the amino acid sequence of SEQ ID NO: 36; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 14; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
37. In an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like
domain of MerTK.
[0227] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 95 and
SEQ ID NO: 76, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a Ig-like domain of
MerTK.
[0228] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-Hl comprising
the amino acid
sequence of SEQ ID NO: 44; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 45;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 46. In one
embodiment, the
antibody comprises (a) HVR-Hl comprising the amino acid sequence of SEQ ID NO:
44; (b) HVR-
H2 comprising the amino acid sequence of SEQ ID NO: 45; and (c) HVR-H3
comprising the amino
acid sequence of SEQ ID NO: 46. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0229] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-Li
comprising the amino acid
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sequence of SEQ ID NO: 41; (b) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 42;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 43. In one
embodiment, the
antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:
41; (b) HVR-
L2 comprising the amino acid sequence of SEQ ID NO: 42; and (c) HVR-L3
comprising the amino
acid sequence of SEQ ID NO: 43. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0230] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 44, (ii) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 45, and (iii) HVR-H3 comprising an amino acid sequence
selected from
SEQ ID NO: 46; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 41, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 43. In an exemplary embodiment, the anti-
MerTK antibody
binds to a Ig-like domain of MerTK.
[0231] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 44; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 45; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 46;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 41; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 42; and (f) HVR-L3 comprising an amino acid
sequence
selected from SEQ ID NO:43. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0232] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins ofinnnunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0233] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
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sequence identity to the amino acid sequence of SEQ ID NO: 96. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 96. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 96, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-Hl
comprising the
amino acid sequence of SEQ ID NO: 44, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO:45, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 46. In
an exemplary
embodiment, the anti-MerTK antibody binds to a Ig-like domain of MerTK.
[0234] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 77. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 77, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 41; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 42; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
43. In an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like
domain of MerTK.
[0235] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 96 and
SEQ ID NO: 77, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a Ig-like domain of
MerTK.
[0236] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-Hl comprising
the amino acid
sequence of SEQ ID NO: 50; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 51;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 52. In one
embodiment, the
antibody comprises (a) HVR-Hl comprising the amino acid sequence of SEQ ID NO:
50; (b) HVR-
H2 comprising the amino acid sequence of SEQ ID NO: 51; and (c) HVR-H3
comprising the amino
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acid sequence of SEQ ID NO: 52. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0237] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-Li
comprising the amino acid
sequence of SEQ ID NO: 47; (b) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 48;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 49. In one
embodiment, the
antibody comprises (a) HVR-Li comprising the amino acid sequence of SEQ ID NO:
47; (b) HVR-
L2 comprising the amino acid sequence of SEQ ID NO: 48; and (c) HVR-L3
comprising the amino
acid sequence of SEQ ID NO: 49. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0238] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 50, (ii) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 51, and (iii) HVR-H3 comprising an amino acid sequence
selected from
SEQ ID NO: 52; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-Li comprising the amino acid sequence of SEQ
ID NO: 47, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 49. In an exemplary embodiment, the anti-
MerTK antibody
binds to a Ig-like domain of MerTK.
[0239] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 50; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 51; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 52;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 48; and (f) HVR-L3 comprising an amino acid
sequence
selected from SEQ ID NO: 49. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0240] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins ofinnnunological Interest, 5th Ed.
Public Health Service,
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National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0241] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 97. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 97. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 97, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 50, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 51, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 52. In
an exemplary
embodiment, the anti-MerTK antibody binds to a Ig-like domain of MerTK.
[0242] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 78. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 78. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 78, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 47; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 48; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
49. In an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like
domain of MerTK.
[0243] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 97 and
SEQ ID NO: 78, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a Ig-like domain of
MerTK.
[0244] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
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sequence identity to the amino acid sequence of SEQ ID NO: 98. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 98. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 98, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 50, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 51, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 52. In
an exemplary
embodiment, the anti-MerTK antibody binds to a Ig-like domain of MerTK.
[0245] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 79. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 79, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 47; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 48; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
49. In an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like
domain of MerTK.
[0246] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 98 and
SEQ ID NO: 79, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a Ig-like domain of
MerTK.
[0247] In another aspect, an anti-MerTK antibody comprises a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 108. In certain embodiments, a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to the
reference sequence, but an anti-
MerTK antibody comprising that sequence retains the ability to bind to MerTK.
In certain
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embodiments, a total of 1 to 10 amino acids have been substituted, inserted
and/or deleted in SEQ ID
NO: 108. In certain embodiments, substitutions, insertions, or deletions occur
in regions outside the
HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody comprises the
heavy chain sequence in
SEQ ID NO: 108, including post-translational modifications of that sequence.
In a particular
embodiment, the heavy chain comprises one, two or three HVRs selected from:
(a) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 50, (b) HVR-H2 comprising the
amino acid
sequence of SEQ ID NO:51, and (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO:52.
In an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like domain
of MerTK.
[0248] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 116. In certain
embodiments, a light
chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity
contains substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the
reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to bind
to MerTK. In certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted
and/or deleted in SEQ ID NO: 116. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the light chain sequence in SEQ ID NO: 116, including post-translational
modifications of that
sequence. In a particular embodiment, the light chain comprises one, two or
three HVRs selected
from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47; (b) HVR-
L2 comprising
the amino acid sequence of SEQ ID NO: 48; and (c) HVR-L3 comprising the amino
acid sequence of
SEQ ID NO: 49. In an exemplary embodiment, the anti-MerTK antibody binds to a
Ig-like domain of
MerTK.
[0249] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
heavy chain as in any of the embodiments provided above, and a light chain as
in any of the
embodiments provided above. In one embodiment, the antibody comprises the
heavy chain and light
chain sequences in SEQ ID NO: 108 and SEQ ID NO: 116, respectively, including
post-translational
modifications of those sequences. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0250] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 99. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 99. In certain embodiments, substitutions, insertions, or
deletions occur in regions
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outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 99, including post-translational modifications of that sequence.
In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 50, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 51, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 52. In
an exemplary
embodiment, the anti-MerTK antibody binds to a Ig-like domain of MerTK.
[0251] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 80. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 80, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 47; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 48; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
49. In an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like
domain of MerTK.
[0252] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 99 and
SEQ ID NO: 80, respectively, including post-translational modifications of
those sequences. In an
exemplary embodiment, the anti-MerTK antibody binds to a Ig-like domain of
MerTK.
[0253] In another aspect, an anti-MerTK antibody comprises a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the
amino acid sequence of SEQ ID NO: 109. In certain embodiments, a heavy chain
sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to the
reference sequence, but an anti-
MerTK antibody comprising that sequence retains the ability to bind to MerTK.
In certain
embodiments, a total of 1 to 10 amino acids have been substituted, inserted
and/or deleted in SEQ ID
NO: 109. In certain embodiments, substitutions, insertions, or deletions occur
in regions outside the
HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody comprises the
heavy chain sequence in
SEQ ID NO: 109, including post-translational modifications of that sequence.
In a particular
embodiment, the heavy chain comprises one, two or three HVRs selected from:
(a) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 50, (b) HVR-H2 comprising the
amino acid
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sequence of SEQ ID NO: 51, and (c) HVR-H3 comprising the amino acid sequence
of SEQ ID NO:
52. In an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like
domain of MerTK.
[0254] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 117. In certain
embodiments, a light
chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity
contains substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the
reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to bind
to MerTK. In certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted
and/or deleted in SEQ ID NO: 117. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the light chain sequence in SEQ ID NO: 117, including post-translational
modifications of that
sequence. In a particular embodiment, the light chain comprises one, two or
three HVRs selected
from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47; (b) HVR-
L2 comprising
the amino acid sequence of SEQ ID NO: 48; and (c) HVR-L3 comprising the amino
acid sequence of
SEQ ID NO: 49. In an exemplary embodiment, the anti-MerTK antibody binds to a
Ig-like domain of
MerTK.
[0255] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
heavy chain as in any of the embodiments provided above, and a light chain as
in any of the
embodiments provided above. In one embodiment, the antibody comprises the
heavy chain and light
chain sequences in SEQ ID NO: 109 and SEQ ID NO: 117, respectively, including
post-translational
modifications of those sequences. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0256] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising
the amino acid
sequence of SEQ ID NO: 56; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 57;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 58. In one
embodiment, the
antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:
56; (b) HVR-
H2 comprising the amino acid sequence of SEQ ID NO: 57; and (c) HVR-H3
comprising the amino
acid sequence of SEQ ID NO: 58. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0257] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-L1
comprising the amino acid
sequence of SEQ ID NO: 53; (b) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 54;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 55. In one
embodiment, the
antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:
53; (b) HVR-
L2 comprising the amino acid sequence of SEQ ID NO: 54; and (c) HVR-L3
comprising the amino
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acid sequence of SEQ ID NO: 55. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0258] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-Hl
comprising the amino acid sequence of SEQ ID NO: 56, (ii) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 57, and (iii) HVR-H3 comprising an amino acid sequence
selected from
SEQ ID NO: 58; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-Li comprising the amino acid sequence of SEQ
ID NO: 53, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 54, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 55. In an exemplary embodiment, the anti-
MerTK antibody
binds to a Ig-like domain of MerTK.
[0259] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 56; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 57; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 58;
(d) HVR-Li comprising the amino acid sequence of SEQ ID NO: 53; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 54; and (f) HVR-L3 comprising an amino acid
sequence
selected from SEQ ID NO: 55. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0260] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins ofinnnunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0261] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 100. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
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In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 100. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 100, including post-translational modifications of that
sequence. In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 56, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 57, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 58. In
an exemplary
embodiment, the anti-MerTK antibody binds to a Ig-like domain of MerTK.
[0262] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 81. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 81. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 81, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-Li
comprising the amino acid sequence of SEQ ID NO: 53; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 54; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
55. In an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like
domain of MerTK.
[0263] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 100
and SEQ ID NO: 81, respectively, including post-translational modifications of
those sequences. In
an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like domain of
MerTK.
[0264] In one aspect, the invention provides an anti-MerTK antibody
comprising at least one, at
least two, or all three VH HVR sequences selected from (a) HVR-Hl comprising
the amino acid
sequence of SEQ ID NO: 62; (b) HVR-H2 comprising the amino acid sequence of
SEQ ID NO: 63;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 64. In one
embodiment, the
antibody comprises (a) HVR-Hl comprising the amino acid sequence of SEQ ID NO:
62; (b) HVR-
H2 comprising the amino acid sequence of SEQ ID NO: 63; and (c) HVR-H3
comprising the amino
acid sequence of SEQ ID NO: 64. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0265] In another aspect, the invention provides an anti-MerTK antibody
comprising at least one,
at least two, or all three VL HVR sequences selected from (a) HVR-Li
comprising the amino acid
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sequence of SEQ ID NO: 59; (b) HVR-L2 comprising the amino acid sequence of
SEQ ID NO: 60;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 61. In one
embodiment, the
antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:
59; (b) HVR-
L2 comprising the amino acid sequence of SEQ ID NO: 60; and (c) HVR-L3
comprising the amino
acid sequence of SEQ ID NO: 61. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0266] In another aspect, an anti-MerTK antibody of the invention comprises
(a) a VH domain
comprising at least one, at least two, or all three VH HVR sequences selected
from (i) HVR-H1
comprising the amino acid sequence of SEQ ID NO: 62, (ii) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 63, and (iii) HVR-H3 comprising an amino acid sequence
selected from
SEQ ID NO: 64; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 59, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO: 60, and (c) HVR-L3
comprising the
amino acid sequence of SEQ ID NO: 61. In an exemplary embodiment, the anti-
MerTK antibody
binds to a Ig-like domain of MerTK.
[0267] In another aspect, the invention provides an anti-MerTK antibody
comprising (a) HVR-
H1 comprising the amino acid sequence of SEQ ID NO: 62; (b) HVR-H2 comprising
the amino acid
sequence of SEQ ID NO: 63; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 64;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59; (e) HVR-L2
comprising the
amino acid sequence of SEQ ID NO: 60; and (f) HVR-L3 comprising an amino acid
sequence
selected from SEQ ID NO: 61. In an exemplary embodiment, the anti-MerTK
antibody binds to a
Ig-like domain of MerTK.
[0268] In any of the above embodiments, an anti-MerTK antibody is
humanized. In one
embodiment, an anti-MerTK antibody comprises HVRs as in any of the above
embodiments, and
further comprises an acceptor human framework, e.g. a human immunoglobulin
framework or a
human consensus framework, optionally with up to 10 amino acid substitutions
(e.g. from 1-2, 1-3, 1-
4, 1-5, 1-6, 1-7, 1-8, 1-9 or 1-10 amino acid substitutions). In exemplary
embodiments, such amino
acid substitutions correspond to the amino acid residues from a rabbit
framework region sequence,
such as, for example, one or more of the following residues: Q2, L4, P43,
and/or F87 in the light
chain variable region framework sequences and/or one or more of the following
residues: V24, 148,
G49, K71, and/or V78 in the heavy chain variable region framework sequences.
The numbering of
amino acid residues is according to the EU numbering system, also called the
EU index, as described
in Kabat et al., Sequences of Proteins ofinnnunological Interest, 5th Ed.
Public Health Service,
National Institutes of Health, Bethesda, MD, 1991. In an exemplary embodiment,
the anti-MerTK
antibody binds to a fibronectin-like domain of MerTK.
[0269] In another aspect, an anti-MerTK antibody comprises a heavy chain
variable domain
(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100%
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sequence identity to the amino acid sequence of SEQ ID NO: 101. In certain
embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains
substitutions (e.g., conservative substitutions), insertions, or deletions
relative to the reference
sequence, but an anti-MerTK antibody comprising that sequence retains the
ability to bind to MerTK.
In certain embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted
in SEQ ID NO: 101. In certain embodiments, substitutions, insertions, or
deletions occur in regions
outside the HVRs (i.e., in the FRs). Optionally, the anti-MerTK antibody
comprises the VH sequence
in SEQ ID NO: 101, including post-translational modifications of that
sequence. In a particular
embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the
amino acid sequence of SEQ ID NO: 62, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID
NO: 63, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 64. In
an exemplary
embodiment, the anti-MerTK antibody binds to a Ig-like domain of MerTK.
[0270] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%,
99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 82. In
certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or
99% identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative
to the reference sequence, but an anti-MerTK antibody comprising that sequence
retains the ability to
bind to MerTK. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted
and/or deleted in SEQ ID NO: 82. In certain embodiments, the substitutions,
insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-
MerTK antibody comprises
the VL sequence in SEQ ID NO: 82, including post-translational modifications
of that sequence. In a
particular embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L 1
comprising the amino acid sequence of SEQ ID NO: 59; (b) HVR-L2 comprising the
amino acid
sequence of SEQ ID NO: 60; and (c) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:
61. In an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like
domain of MerTK.
[0271] In another aspect, an anti-MerTK antibody is provided, wherein the
antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the
embodiments provided
above. In one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO: 101
and SEQ ID NO: 82, respectively, including post-translational modifications of
those sequences. In
an exemplary embodiment, the anti-MerTK antibody binds to a Ig-like domain of
MerTK.
[0272] In a further aspect, the invention provides an antibody that
competes for binding to
MerTK with an anti-MerTK reference antibody provided herein. For example, in
certain
embodiments, an antibody is provided that competes for binding to MerTK with
one or more of the
following anti-MerTK reference antibodies: an antibody comprising a VH
comprising the amino acid
sequence of SEQ ID NO: 83 and a VL comprising the amino acid sequence of SEQ
ID NO: 65; an
antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 84
and a VL
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comprising the amino acid sequence of SEQ ID NO: 66; an antibody comprising a
VH comprising the
amino acid sequence of SEQ ID NO: 85 and a VL comprising the amino acid
sequence of SEQ ID
NO: 67; an antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO:
102 and a light chain comprising the amino acid sequence of SEQ ID NO: 110; an
antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL
comprising the
amino acid sequence of SEQ ID NO: 68; an antibody comprising a heavy chain
comprising the amino
acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid
sequence of SEQ ID
NO: 111; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 87 and a
VL comprising the amino acid sequence of SEQ ID NO: 69; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 88 and a VL comprising the amino acid
sequence of SEQ ID
NO: 70; an antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO:
104 and a light chain comprising the amino acid sequence of SEQ ID NO: 112; an
antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO: 89 and a VL
comprising the
amino acid sequence of SEQ ID NO: 70; an antibody comprising a heavy chain
comprising the amino
acid sequence of SEQ ID NO: 105 and a light chain comprising the amino acid
sequence of SEQ ID
NO: 113; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 90 and a
VL comprising the amino acid sequence of SEQ ID NO: 71; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 91 and a VL comprising the amino acid
sequence of SEQ ID
NO: 72; an antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO:
106 and a light chain comprising the amino acid sequence of SEQ ID NO: 114; an
antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO: 92 and a VL
comprising the
amino acid sequence of SEQ ID NO: 73; an antibody comprising a heavy chain
comprising the amino
acid sequence of SEQ ID NO: 107 and a light chain comprising the amino acid
sequence of SEQ ID
NO: 115; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 93 and a
VL comprising the amino acid sequence of SEQ ID NO: 74; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 94 and a VL comprising the amino acid
sequence of SEQ ID
NO: 75; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 95 and a
VL comprising the amino acid sequence of SEQ ID NO: 76; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 96 and a VL comprising the amino acid
sequence of SEQ ID
NO: 77; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 97 and a
VL comprising the amino acid sequence of SEQ ID NO: 78; an antibody comprising
a VH comprising
the amino acid sequence of SEQ ID NO: 98 and a VL comprising the amino acid
sequence of SEQ ID
NO: 79; an antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO:
108 and a light chain comprising the amino acid sequence of SEQ ID NO: 116; an
antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO: 99 and a VL
comprising the
amino acid sequence of SEQ ID NO: 80; an antibody comprising a heavy chain
comprising the amino
acid sequence of SEQ ID NO: 109 and a light chain comprising the amino acid
sequence of SEQ ID
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NO: 117; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 100 and
a VL comprising the amino acid sequence of SEQ ID NO: 81; and an antibody
comprising a VH
comprising the amino acid sequence of SEQ ID NO: 101 and a VL comprising the
amino acid
sequence of SEQ ID NO: 82. In some embodiments, the isolated antibody binds to
human MerTK.
In some embodiments, the reference antibody is Y323, which is commercially
available (abeam
catalog no. ab52968).
[0273] In a
further aspect, the invention provides an antibody that binds to the same
epitope as an
anti-MerTK antibody provided herein. For example, in certain embodiments, an
antibody is provided
that binds to the same epitope as any one of the following anti-MerTK
antibodies: an antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO: 83 and a VL
comprising the
amino acid sequence of SEQ ID NO: 65; an antibody comprising a VH comprising
the amino acid
sequence of SEQ ID NO: 84 and a VL comprising the amino acid sequence of SEQ
ID NO: 66; an
antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 85
and a VL
comprising the amino acid sequence of SEQ ID NO: 67; an antibody comprising a
heavy chain
comprising the amino acid sequence of SEQ ID NO: 102 and a light chain
comprising the amino acid
sequence of SEQ ID NO: 110; an antibody comprising a VH comprising the amino
acid sequence of
SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 68; an
antibody
comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 103
and a light chain
comprising the amino acid sequence of SEQ ID NO: 111; an antibody comprising a
VH comprising
the amino acid sequence of SEQ ID NO: 87 and a VL comprising the amino acid
sequence of SEQ ID
NO: 69; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 88 and a
VL comprising the amino acid sequence of SEQ ID NO: 70; an antibody comprising
a heavy chain
comprising the amino acid sequence of SEQ ID NO: 104 and a light chain
comprising the amino acid
sequence of SEQ ID NO: 112; an antibody comprising a VH comprising the amino
acid sequence of
SEQ ID NO: 89 and a VL comprising the amino acid sequence of SEQ ID NO: 70; an
antibody
comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 105
and a light chain
comprising the amino acid sequence of SEQ ID NO: 113; an antibody comprising a
VH comprising
the amino acid sequence of SEQ ID NO: 90 and a VL comprising the amino acid
sequence of SEQ ID
NO: 71; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 91 and a
VL comprising the amino acid sequence of SEQ ID NO: 72; an antibody comprising
a heavy chain
comprising the amino acid sequence of SEQ ID NO: 106 and a light chain
comprising the amino acid
sequence of SEQ ID NO: 114; an antibody comprising a VH comprising the amino
acid sequence of
SEQ ID NO: 92 and a VL comprising the amino acid sequence of SEQ ID NO: 73; an
antibody
comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 107
and a light chain
comprising the amino acid sequence of SEQ ID NO: 115; an antibody comprising a
VH comprising
the amino acid sequence of SEQ ID NO: 93 and a VL comprising the amino acid
sequence of SEQ ID
NO: 74; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 94 and a
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VL comprising the amino acid sequence of SEQ ID NO: 75. In certain
embodiments, an antibody is
provided that binds to an epitope within an Fibronectin-like domain of MerTK
consisting of amino
acid residues 286-384 or 388-480 of MerTK SEQ ID NO: 129. In some embodiments,
the antibody
binds to the same epitope as antibody is Y323, which is commercially available
(abcam catalog no.
ab52968).
[0274] In a further aspect, the invention provides an antibody that binds
to the same epitope as an
anti-MerTK antibody provided herein. For example, in certain embodiments, an
antibody is provided
that binds to the same epitope as any one of the following anti-MerTK
antibodies: an antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL
comprising the
amino acid sequence of SEQ ID NO: 76; an antibody comprising a VH comprising
the amino acid
sequence of SEQ ID NO: 96 and a VL comprising the amino acid sequence of SEQ
ID NO: 77; an
antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 97
and a VL
comprising the amino acid sequence of SEQ ID NO: 78; an antibody comprising a
VH comprising the
amino acid sequence of SEQ ID NO: 98 and a VL comprising the amino acid
sequence of SEQ ID
NO: 79; an antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO:
108 and a light chain comprising the amino acid sequence of SEQ ID NO: 116; an
antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO: 99 and a VL
comprising the
amino acid sequence of SEQ ID NO: 80; an antibody comprising a heavy chain
comprising the amino
acid sequence of SEQ ID NO: 109 and a light chain comprising the amino acid
sequence of SEQ ID
NO: 117; an antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO: 100 and
a VL comprising the amino acid sequence of SEQ ID NO: 81; and an antibody
comprising a VH
comprising the amino acid sequence of SEQ ID NO: 101 and a VL comprising the
amino acid
sequence of SEQ ID NO: 82. In certain embodiments, an antibody is provided
that binds to an
epitope within an Ig-like domain of MerTK consisting of amino acid residues 76-
195 or 199-283 of
MerTK SEQ ID NO: 129.
[0275] In a further aspect of the invention, an anti-MerTK antibody
according to any of the
above embodiments is a monoclonal antibody, including a chimeric, humanized or
human antibody.
In one embodiment, an anti-MerTK antibody is an antibody fragment, e.g., a Fv,
Fab, Fab', scFv,
diabody, or F(ab')2. fragment. In another embodiment, the antibody is a full
length antibody, e.g., an
intact IgG1 antibody or other antibody class or isotype as defined herein. In
certain embodiments, the
antibody comprises a mutation in the Fc region that reduces binding to Fc
receptors and/or
complement. In one embodiment, the antibody comprises a LALAPG mutation in the
Fc region.
[0276] In a further aspect, an anti-MerTK antibody according to any of the
above embodiments
may incorporate any of the features, singly or in combination, as described in
Sections 1-8 below:
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1. MerTK Biological Activity
[0277] In some embodiments, the antibodies reduce MerTK mediated clearance
of apoptotic
cells by phagocytes, e.g., the clearance of apoptotic cells is reduced by 1-10
fold, 1-8 fold, 1-5 fold, 1-
4 fold, 1-3 fold, 1-2 fold, 2-10 fold, 2-8 fold, 2-5 fold, 2-4 fold, 2-3 fold,
3-10 fold, 3-8 fold, 3-5 fold,
3-4 fold, or by about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6
fold, 1.7 fold, 1.8 fold, 1.9 fold,
2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7
fold, 2.8 fold, 2.9 fold, 3.0 fold, 3.1
fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold,
3.9 fold, 4.0 fold, 4.1 fold, 4.2
fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9 fold,
5.0 fold, 5.1 fold, 5.2 fold, 5.3
fold, 5.4 fold, 5.5 fold, 5.6 fold, 5.7 fold, 5.8 fold, 5.9 fold, 6.0 fold,
6.1 fold, 6.2 fold, 6.3 fold, 6.4
fold, 6.5 fold, 6.6 fold, 6.7 fold, 6.8 fold, 6.9 fold, 7.0 fold, 7.1 fold,
7.2 fold, 7.3 fold, 7.4 fold, 7.5
fold, 7.6 fold, 7.7 fold, 7.8 fold, 7.9 fold, or 8.0 fold. In some
embodiments, the phagocytes are
macrophages. In some such embodiments, the macrophages are tumor-associated
macrophages
(TAMs). In humans, TAMs may be identified based on expression of various cell-
surface markers,
including CD14, HLA-DR (MHC class II), CD312, CD115, CD16, CD163, CD204,
CD206, and
CD301. Furthermore, the production of specific functional biomarkers, such as
matrix
metalloproteinases, IL-10, inducible nitric oxide synthase (iNOS), TNF-alpha,
or IL-12 may be
combined with cell-surface biomarkers to accurately identify TAM populations
(Quatromoni, J., et
al., Am J Transl Res. 4 (2012): 376-389.) The clearance of apoptotic cells may
be measured by any
assay known to one of skill in the art for such purpose. For example, for in
vitro apoptotic cell
clearance assays, phagocytes such as mouse peritoneal macrophages or human
monocyte derived
macrophages are used. Apoptotic cells are generated by treatment with
dexamethasone and labeled
with a detection probe. Phagocytosis can be analyzed by microscopy or flow
cytometry after
incubation apoptotic cells with phagocytes. In some embodiments, the clearance
of apoptotic cells is
reduced as measured in such an apoptotic cell clearance assay at room
temperature. For example, for
in vivo apoptotic clearance assays, mice are injected with dexamethasone to
induce thymocyte death.
Resident macrophages in the thymus recognize and engulf the dying/dead cells
(Seitz, H. M. J
Immunol. 178(9) 5635-5642 (2007). In some embodiments, the clearance of
apoptotic cells is
reduced as measured in such an apoptotic cell clearance assay in vivo. In some
embodiments, the
antibodies reduce ligand-mediated MerTK signaling. In some embodiments, the
ligand is hGAS6-Fc
(EC50 = ¨ 84 pM). In some embodiments, the antibodies induce a pro-
inflammatory response. In
some emobidments, the antibodies induce a type I IFN response.
[0278] In some embodiments, an anti-MerTK antibody of the present
disclosure reduces
phagocytic activity of apoptotic cells by about 10-100%, 20-100%, 30-100%, 40-
100%, 50-100%, 60-
100%, 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, 95-100%, 10-95%, 20-95%, 30-
95%, 40-
95%, 50-95%, 60-95%, 70-95%, 75-95%, 80-95%, 85-95%, 90-95%, 10-90%, 20-90%,
30-90%, 40-
90%, 50-90%, 60-90%, 70-90%, 75-90%, 80-90%, 85-90%, 10-85%, 20-85%, 30-85%,
40-85%, 50-
85%, 60-85%, 70-85%, 75-85%, 80-85%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%,
60-80%, 70-
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80%, 75-80%, 10-75%, 20-75%, 30-75%, 40-75%, 50-75%, 60-75%, 70-75%, 10-70%,
20-70%, 30-
70%, 40-70%, 50-70%, 60-70%, 10-65%, 20-65%, 30-65%, 40-65%, 50-65%, 60-65%,
10-60%, 20-
60%, 30-60%, 40-60%, 50-60%, 10-55%, 20-55%, 30-55%, 40-55%, 50-55%, 10-40%,
20-40%, or
30-40%, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%,
85%, 90%,
95%, 96%, 97%, 98% or 99%. In some embodiments, the anti-MerTK antibody has a
half maximal
inhibitory concentration (IC50) for reducing phagocytic activity of apoptotic
cells of about 1 pM ¨ 50
pM, 1 pM - 100 pM, 1 pM ¨500 pM, 1 pM ¨ 1 nM, 1 pM ¨ 1.5 nM, 5 pM¨SO pM, 5 pM -
100 pM, 5
pM ¨ 500 pM, 5 pM ¨ 1 nM, 5 pM ¨ 1.5 nM, 10 pM ¨ 50 pM, 10 pM - 100 pM, 10 pM
¨ 500 pM, 10
pM ¨ 1 nM, 10 pM ¨ 1.5 nM, 50 pM - 100 pM, 50 pM ¨ 500 pM, 50 pM ¨ 1 nM, 50 pM
¨ 1.5 nM,
100 pM ¨500 pM, 100 pM ¨ 1 nM, or 100 pM ¨ 1.5 nM. Exemplary methods for
determining
phagocytic activity and IC50 are described in the Examples herein below.
[0279] In some embodiments, an anti-MerTK antibody of the present
disclosure enhances the
activity of a checkpoint inhibitor by about 1-2 fold, 1-5 fold, 1-10 fold, 1-
15 fold, 1-20 fold, 1-25 fold,
1-30 fold, 1-50 fold, 1-75 fold, 1-100 fold, 1-150 fold, 1-200 fold, 1-250
fold, 1.5-2 fold, 1.5-5 fold,
1.5-10 fold, 1.5-15 fold, 1.5-20 fold, 1.5-25 fold, 1.5-30 fold, 1.5-50 fold,
1.5-75 fold, 1.5-100 fold,
1.5-150 fold, 1.5-200 fold, 1.5-250 fold, 2-5 fold, 2-10 fold, 2-15 fold, 2-20
fold, 2-25 fold, 2-30 fold,
2-50 fold, 2-75 fold, 2-100 fold, 2-150 fold, 2-200 fold, 2-250 fold, 2.5-5
fold, 2.5-10 fold, 2.5-15
fold, 2.5-20 fold, 2.5-25 fold, 2.5-30 fold, 2.5-50 fold, 2.5-75 fold, 2.5-100
fold, 2.5-150 fold, 2.5-200
fold, 2.5-250 fold, 5-10 fold, 5-15 fold, 5-20 fold, 5-25 fold, 5-30 fold, 5-
50 fold, 5-75 fold, 5-100
fold, 5-150 fold, 5-200 fold, 5-250 fold, 10-15 fold, 10-20 fold, 10-25 fold,
10-30 fold, 10-50 fold, 10-
75 fold, 10-100 fold, 10-150 fold, 10-200 fold, 10-250 fold, 20-25 fold, 20-30
fold, 20-50 fold, 20-75
fold, 20-100 fold, 20-150 fold, 20-200 fold, 20-250 fold, 25-30 fold, 25-50
fold, 25-75 fold, 25-100
fold, 25-150 fold, 25-200 fold, or 25-250 fold or by at least about 1 fold, 2
fold, 5 fold, 10 fold, 15
fold 20 fold 25 fold, 30 fold, 40 fold, 50 fold 60 fold, 70 fold, 75 fold, 80
fold, 90 fold, 100 fold, 125
fold, 150 fold, 200 fold, 225 fold or 250 fold. In certain embodiments, an
anti-MerTK antibody of the
present disclosure enhances the activity of a checkpoint inhibitor as
determined using an assay as
described in the Examples herein below, such as, for example, by determining a
reduction in tumor
volume in a mouse tumor model using a combination of an anti-MerTK antibody
plus a checkpoint
inhibitor as compared to the reduction in tumor volume using the checkpoint
inhibitor alone. In
certain embodiments, the reduction in tumor volume is determined after at
least 10 days, 14 days, 20
days, 21 days or 30 days after treatment with the therapeutic agents. In
certain embodiments, the
checkpoint inhibitor is a anti-PD1 axis antagonist. In one exemplary
embodiment, the checkpoint
inhibitor is an anti-PD-Li antibody. In another embodiment, the checkpoint
inhibitor is an anti-PD1
antibody
[0280] In some embodiments, an anti-MerTK antibody of the present
disclosure increases cell-
free DNA (cfDNA) and/or circulating tumor DNA (ctDNA), e.g., in a blood or
plasma sample, by
about 1-2 fold, 1-3 fold, 1-4 fold, 1-5 fold, 1-10 fold, 1.5-2 fold, 1.5-3
fold, 1.5-4 fold, 1.5-5 fold, 1.5-
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fold, 2-3 fold, 2-4 fold, 2-5 fold, 2-10 fold, 3-5 fold, 3-10 fold, 4-5 fold,
4-10 fold, 5-10 fold, or by
at least about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, or 10 fold. In certain
embodiments, an anti-MerTK
antibody of the present disclosure increases cell-free DNA (cfDNA) and/or
circulating tumor DNA
(ctDNA) as determined using an assay as described in the Examples herein
below, such as, for
example, by isolating cfDNA and/or ctDNA from a blood or plasma sample and
detecting levels of
cfDNA and/or ctDNA using PCR and quantitative DNA electrophoresis.
2. Antibody Affinity & Specificity
[0281] In certain embodiments, an anti-MerTK antibody provided herein has a
dissociation
constant (Kd) of < 1 M, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or <
0.001 nM, or about
1 pM ¨ 0.1 nM, 1 pM ¨ 0.2 nM, 1 pM ¨ 0.5 nM, 1 pM ¨ 1 nM, 1 pM -2 nM, 1 pM ¨ 5
nM, 1 pM ¨ 10
nM, 1 pM ¨ 15 nM, 5 pM ¨ 0.1 nM, 5 pM ¨ 0.2 nM, 5 pM ¨ 0.5 nM, 5 pM ¨ 1 nM, 5
pM -2 nM, 5 pM
¨ 5 nM, 5 pM ¨ 10 nM, 5 pM ¨ 15 nM, 10 pM ¨ 0.1 nM, 10 pM ¨ 0.2 nM, 10 pM ¨
0.5 nM, 10 pM ¨
1 nM, 10 pM -2 nM, 10 pM ¨ 5 nM, 10 pM ¨ 10 nM, 10 pM ¨ 15 nM, 20 pM ¨ 0.1 nM,
20 pM ¨ 0.2
nM, 20 pM ¨ 0.5 nM, 20 pM ¨ 1 nM, 20 pM -2 nM, 20 pM ¨5 nM, 20 pM ¨ 10 nM, 20
pM ¨ 15 nM,
25 pM ¨0.1 nM, 25 pM ¨0.2 nM, 25 pM ¨0.5 nM, 25 pM ¨ 1 nM, 25 pM -2 nM, 25 pM
¨ 5 nM, 25
pM¨ 10 nM, 25 pM¨ 15 nM, 50 pM ¨ 0.1 nM, 50 pM ¨ 0.2 nM, 50 pM ¨ 0.5 nM, 50 pM
¨ 1 nM, 50
pM -2 nM, 50 pM ¨ 5 nM, 50 pM ¨ 10 nM, 50 pM¨ 15 nM, 100 pM ¨ 0.2 nM, 100 pM ¨
0.5 nM, 100
pM ¨ 1 nM, 100 pM -2 nM, 100 pM ¨ 5 nM, 100 pM ¨ 10 nM, or 100 pM ¨ 15 nM. In
certain
embodiments, the Kd of the anti-MerTK antibody as disclosed herein is measured
at 25 C. In certain
embodiments, the Kd of the anti-MerTK antibody as disclosed herein is measured
at 37 C.
[0282] In one embodiment, Kd is measured by a radiolabeled antigen binding
assay (RIA). In
one embodiment, an RIA is performed with the Fab version of an antibody of
interest and its antigen.
For example, solution binding affinity of Fabs for antigen is measured by
equilibrating Fab with a
minimal concentration of ('25I)-labeled antigen in the presence of a titration
series of unlabeled
antigen, then capturing bound antigen with an anti-Fab antibody-coated plate
(see, e.g., Chen et al., J.
Mol. Biol. 293:865-881(1999)). To establish conditions for the assay,
MICROTITER multi-well
plates (Thermo Scientific) are coated overnight with 5 g/m1 of a capturing
anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2%
(w/v) bovine serum
albumin in PBS for two to five hours at room temperature (approximately 23 C).
In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM ['25I-antigen are mixed with serial
dilutions of a Fab of
interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12,
in Presta et al., Cancer
Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight;
however, the incubation
may continue for a longer period (e.g., about 65 hours) to ensure that
equilibrium is reached.
Thereafter, the mixtures are transferred to the capture plate for incubation
at room temperature (e.g.,
for one hour). The solution is then removed and the plate washed eight times
with 0.1% polysorbate
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20 (TWEEN-20 ) in PBS. When the plates have dried, 150 id/well of scintillant
(MICROSCINT-20
Tm; Packard) is added, and the plates are counted on a TOPCOUNT TM gamma
counter (Packard) for
ten minutes. Concentrations of each Fab that give less than or equal to 20% of
maximal binding are
chosen for use in competitive binding assays.
[0283] According to another embodiment, Kd is measured using a BIACORE
surface plasmon
resonance assay. For example, an assay using a BIACORE -2000 or a BIACORE -
3000 (BIAcore,
Inc., Piscataway, NJ) is performed at 25 C with immobilized antigen CMS chips
at ¨10 response units
(RU). In one embodiment, carboxymethylated dextran biosensor chips (CMS,
BIACORE, Inc.) are
activated with N-ethyl-N'- (3-dimethylaminopropy1)-carbodiimide hydrochloride
(EDC) and N-
hydroxy succinimide (NHS) according to the supplier's instructions. Antigen is
diluted with 10 mM
sodium acetate, pH 4.8, to 5 itg/m1 (-0.2 iiM) before injection at a flow rate
of 5 id/minute to achieve
approximately 10 response units (RU) of coupled protein. Following the
injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics measurements,
two-fold serial
dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%
polysorbate 20 (TWEEN-20Tm)
surfactant (PBST) at 25 C at a flow rate of approximately 25 id/min.
Association rates (kon) and
dissociation rates (koff) are calculated using a simple one-to-one Langmuir
binding model
(BIACORE Evaluation Software version 3.2) by simultaneously fitting the
association and
dissociation sensorgrams. The equilibrium dissociation constant (Kd) is
calculated as the ratio
koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-
rate exceeds 106W' 54
by the surface plasmon resonance assay above, then the on-rate can be
determined by using a
fluorescent quenching technique that measures the increase or decrease in
fluorescence emission
intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25 C of
a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as
measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv
Instruments) or a
8000-series SLM-AMINCO TM spectrophotometer (ThermoSpectronic) with a stirred
cuvette.
[0284] In certain embodiments, an anti-MerTK antibody as disclosed herein
binds to one or more
of human MerTK, cyno MerTK, mouse MerTK and/or rat MerTK. In one embodiment,
an anti-
MerTK antibody as disclosed herein binds specifically to human MerTK. In one
embodiment, an
anti-MerTK antibody as disclosed herein binds to human MerTK and cyno MerTK.
In one
embodiment, an anti-MerTK antibody as disclosed herein binds to human MerTK
and mouse MerTK.
In one embodiment, an anti-MerTK antibody as disclosed herein binds to human
MerTK, cyno
MerTK and mouse MerTK. In one embodiment, an anti-MerTK antibody as disclosed
herein binds to
human MerTK, cyno MerTK, mouse MerTK and rat MerTK. In one embodiment, an anti-
MerTK
antibody as disclosed herein binds specifically to mouse MerTK.
[0285] In certain embodiments, an anti-MerTK antibody as disclosed herein
binds to an Ig-like
domain of MerTK. In one embodiment, an anti-MerTK antibody that binds to an Ig-
like domain of
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MerTK binds to one or more amino acid residues in the Ig-like domain
corresponding to amino acid
residues 76-195 of MerTK SEQ ID NO: 129, e.g., the anti-MerTK antibody binds
to at least 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 amino acids or 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or
1-10 amino acid residues of
residues 76-195 of MerTK SEQ ID NO: 129. In one embodiment, an anti-MerTK
antibody that binds
to an Ig-like domain of MerTK binds to one or more amino acid residues in the
Ig-like domain
corresponding to amino acid residues 199-283 of MerTK SEQ ID NO: 129, e.g.,
the anti-MerTK
antibody binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids or 1-
2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,
1-9, or 1-10 amino acid residues of residues 199-283 of MerTK SEQ ID NO: 129.
[0286] In certain embodiments, an anti-MerTK antibody as disclosed herein
binds to a
fibronectin-like domain of MerTK. In one embodiment, an anti-MerTK antibody
that binds to an
fibronectin-like domain of MerTK binds to one or more amino acid residues in
the fibronectin-like
domain corresponding to amino acid residues 286-384 of MerTK SEQ ID NO: 129,
e.g., the anti-
MerTK antibody binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids
or 1-2, 1-3, 1-4, 1-5, 1-6, 1-
7, 1-8, 1-9, or 1-10 amino acid residues of residues 286-384 of MerTK SEQ ID
NO: 129. In one
embodiment, an anti-MerTK antibody that binds to a fibronectin-like domain of
MerTK binds to one
or more amino acid residues in the fibronectin-like domain corresponding to
amino acid residues 388-
480 of MerTK SEQ ID NO: 129, e.g., the anti-MerTK antibody binds to at least
1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 amino acids or 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 amino
acid residues of residues 388-
480 of MerTK SEQ ID NO: 129.
[0287] In an exemplary embodiment, an anti-MerTK antibody as disclosed
herein binds to an Ig-
like domain of human and cyno MerTK. In one embodiment, such an antibody binds
to human and
cyno MerTK with a Kd at 37 C that is approximately the same, e.g., the
antibody binds to cyno
MerTK at 37 C with a Kd that is not more than 10%, 15% or 20% different than
the Kd of the
antibody at 37 C for human MerTK. In certain embodiments, such an antibody
binds to human and
cyno MerTK with a Kd at 37 C that is at least 20 fold, 25 fold or 50 fold
better than the Kd of the
antibody at 37 C for mouse and rat MerTK.
3. Antibody Fragments
[0288] In certain embodiments, an anti-MerTK antibody provided herein is an
antibody
fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-
SH, F(ab')2, Fv, and
scFv fragments, and other fragments described below. For a review of certain
antibody fragments,
see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments,
see, e.g., Pluckthiln, in
The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag,
New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos.
5,571,894 and
5,587,458. For discussion of Fab and F(ab1)2 fragments comprising salvage
receptor binding epitope
residues and having increased in vivo half-life, see U.S. Patent No.
5,869,046.
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[0289] Diabodies are antibody fragments with two antigen-binding sites that
may be bivalent or
bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat.
Med. 9:129-134
(2003); and Hollinger et al., Proc. Natl. Acad. Sc!. USA 90: 6444-6448 (1993).
Triabodies and
tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
[0290] Single-domain antibodies are antibody fragments comprising all or a
portion of the heavy
chain variable domain or all or a portion of the light chain variable domain
of an antibody. In certain
embodiments, a single-domain antibody is a human single-domain antibody
(Domantis, Inc.,
Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).
[0291] Antibody fragments can be made by various techniques, including but
not limited to
proteolytic digestion of an intact antibody as well as production by
recombinant host cells (e.g. E. colt
or phage), as described herein.
4. Chimeric and Humanized Antibodies
[0292] In certain embodiments, an anti-MerTK antibody provided herein is a
chimeric antibody.
Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567;
and Morrison et al.,
Proc. Natl. Acad. Sc!. USA, 81:6851-6855 (1984)). In one example, a chimeric
antibody comprises a
non-human variable region (e.g., a variable region derived from a mouse, rat,
hamster, rabbit, or non-
human primate, such as a monkey) and a human constant region. In a further
example, a chimeric
antibody is a "class switched" antibody in which the class or subclass has
been changed from that of
the parent antibody. Chimeric antibodies include antigen-binding fragments
thereof
[0293] In certain embodiments, a chimeric antibody is a humanized antibody.
Typically, a non-
human antibody is humanized to reduce immunogenicity to humans, while
retaining the specificity
and affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or
more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are
derived from a non-
human antibody, and FRs (or portions thereof) are derived from human antibody
sequences. A
humanized antibody optionally will also comprise at least a portion of a human
constant region. In
some embodiments, some FR residues in a humanized antibody are substituted
with corresponding
residues from a non-human antibody (e.g., the antibody from which the HVR
residues are derived),
e.g., to restore or improve antibody specificity or affinity.
[0294] Humanized antibodies and methods of making them are reviewed, e.g.,
in Almagro and
Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g.,
in Riechmann et al.,
Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sc!. USA 86:10029-
10033 (1989); US
Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal.,
Methods 36:25-34
(2005) (describing specificity determining region (SDR) grafting); Padlan,
Mol. Immunol. 28:489-498
(1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005)
(describing "FR
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shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al.,
Br. J. Cancer, 83:252-
260 (2000) (describing the "guided selection" approach to FR shuffling).
[0295] Human framework regions that may be used for humanization include
but are not limited
to: framework regions selected using the "best-fit" method (see, e.g., Sims et
al. J. Immunol.
151:2296 (1993)); framework regions derived from the consensus sequence of
human antibodies of a
particular subgroup of light or heavy chain variable regions (see, e.g.,
Carter et al. Proc. Natl. Acad.
Sc!. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993));
human mature
(somatically mutated) framework regions or human germline framework regions
(see, e.g., Almagro
and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions
derived from screening
FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997)
and Rosok et al., J. Biol.
Chem. 271:22611-22618 (1996)).
5. Human Antibodies
[0296] In certain embodiments, an anti-MerTK antibody provided herein is a
human antibody.
Human antibodies can be produced using various techniques known in the art.
Human antibodies are
described generally in van Dijk and van de Winkel, Curr. Op/n. Pharmacol. 5:
368-74 (2001) and
Lonberg, Curr. Op/n. Immunol. 20:450-459 (2008).
[0297] Human antibodies may be prepared by administering an immunogen to a
transgenic
animal that has been modified to produce intact human antibodies or intact
antibodies with human
variable regions in response to antigenic challenge. Such animals typically
contain all or a portion of
the human immunoglobulin loci, which replace the endogenous immunoglobulin
loci, or which are
present extrachromosomally or integrated randomly into the animal's
chromosomes. In such
transgenic mice, the endogenous immunoglobulin loci have generally been
inactivated. For review of
methods for obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech.
23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584
describing
XENOMOUSE' technology; U.S. Patent No. 5,770,429 describing HuMAB 0
technology; U.S.
Patent No. 7,041,870 describing K-M MOUSE technology, and U.S. Patent
Application Publication
No. US 2007/0061900, describing VELoCiMouSEO technology). Human variable
regions from intact
antibodies generated by such animals may be further modified, e.g., by
combining with a different
human constant region.
[0298] Human antibodies can also be made by hybridoma-based methods. Human
myeloma and
mouse-human heteromyeloma cell lines for the production of human monoclonal
antibodies have
been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et
al., Monoclonal
Ant/body Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987);
and Boerner et al., J. Immunol., 147: 86 (1991).) Haman antibodies generated
via human B-cell
biidoina technology are aiso described in Li et al., Proc. Natl. Acad Sci.
USA, 103:3557-3562
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(2006). Additional methods include those described, for example, in U.S.
Patent No. 7,189,826
(describing production of monoclonal human IgM antibodies from hybridoma cell
lines) and Ni,
Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas).
Human
hybridoma technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology
and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods
and Findings in
Experimental and Clinical Pharmacology, 27(3):185-91 (2005).
[0299] Human antibodies may also be generated by isolating Fv clone
variable domain
sequences selected from human-derived phage display libraries. Such variable
domain sequences
may then be combined with a desired human constant domain. Techniques for
selecting human
antibodies from antibody libraries are described below.
6. Library-Derived Antibodies
[0300] Anti-MerTK antibodies of the invention may be isolated by screening
combinatorial
libraries for antibodies with the desired activity or activities. For example,
a variety of methods are
known in the art for generating phage display libraries and screening such
libraries for antibodies
possessing the desired binding characteristics. Such methods are reviewed,
e.g., in Hoogenboom et
al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human
Press, Totowa, NJ, 2001)
and further described, e.g., in the McCafferty et al., Nature 348:552-554;
Clackson et al., Nature 352:
624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in
Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu
et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);
Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
Methods 284(1-2): 119-
132(2004).
[0301] In certain phage display methods, repertoires of VH and VL genes are
separately cloned
by polymerase chain reaction (PCR) and recombined randomly in phage libraries,
which can then be
screened for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455
(1994). Phage typically display antibody fragments, either as single-chain Fv
(scFv) fragments or as
Fab fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen
without the requirement of constructing hybridomas. Alternatively, the naive
repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a wide range of
non-self and also self
antigens without any immunization as described by Griffiths et al., EtVIBO J,
12: 725-734 (1993).
Finally, naive libraries can also be made synthetically by cloning
unrearranged V-gene segments from
stem cells, and using PCR primers containing random sequence to encode the
highly variable CDR3
regions and to accomplish rearrangement in vitro, as described by Hoogenboom
and Winter, J. Mol.
Biol., 227: 381-388 (1992). Patent publications describing human antibody
phage libraries include,
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for example: US Patent No. 5,750,373, and US Patent Publication Nos.
2005/0079574, 2005/0119455,
2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0302] Antibodies or antibody fragments isolated from human antibody
libraries are considered
human antibodies or human antibody fragments herein.
7. Multispecific Antibodies
[0303] In certain embodiments, an anti-MerTK antibody provided herein is a
multispecific
antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal
antibodies that have
binding specificities for at least two different sites. In certain
embodiments, one of the binding
specificities is for MerTK and the other is for any other antigen. In certain
embodiments, bispecific
antibodies may bind to two different epitopes of MerTK. Bispecific antibodies
may also be used to
localize cytotoxic agents to cells which express MerTK. Bispecific antibodies
can be prepared as full
length antibodies or antibody fragments.
[0304] Techniques for making multispecific antibodies include, but are not
limited to,
recombinant co-expression of two immunoglobulin heavy chain-light chain pairs
having different
specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829,
and Traunecker et al.,
EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S.
Patent No. 5,731,168).
Multi-specific antibodies may also be made by engineering electrostatic
steering effects for making
antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or
more antibodies or
fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science,
229: 81(1985)); using
leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al.,
J. Immunol., 148(5):1547-
1553 (1992)); using "diabody" technology for making bispecific antibody
fragments (see, e.g.,
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using
single-chain Fv (sFv)
dimers (see,e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing
trispecific antibodies as
described, e.g., in Tat et al. J. Immunol. 147: 60 (1991).
[0305] Engineered antibodies with three or more functional antigen binding
sites, including
"Octopus antibodies," are also included herein (see, e.g. US 2006/0025576A1).
[0306] The antibody or fragment herein also includes a "Dual Acting FAb" or
"DAF"
comprising an antigen binding site that binds to MerTK as well as another,
different antigen (see,
US 2008/0069820, for example).
8. Antibody Variants
[0307] In certain embodiments, amino acid sequence variants of the anti-
MerTK antibody
provided herein are contemplated. For example, it may be desirable to improve
the binding affinity
and/or other biological properties of the anti-MerTK antibody. Amino acid
sequence variants of an
antibody may be prepared by introducing appropriate modifications into the
nucleotide sequence
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encoding the antibody, or by peptide synthesis. Such modifications include,
for example, deletions
from, and/or insertions into and/or substitutions of residues within the amino
acid sequences of the
antibody. Any combination of deletion, insertion, and substitution can be made
to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
a) Substitution, Insertion, and Deletion Variants
[0308] In certain embodiments, antibody variants having one or more amino
acid substitutions
are provided. Sites of interest for substitutional mutagenesis include the
HVRs and FRs.
Conservative substitutions are shown in Table 1 under the heading of
"preferred substitutions." More
substantial changes are provided in Table 1 under the heading of "exemplary
substitutions," and as
further described below in reference to amino acid side chain classes. Amino
acid substitutions may
be introduced into an antibody of interest and the products screened for a
desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or improved ADCC
or CDC.
TABLE 1
Original Exemplary
Preferred
Residue Substitutions
Substitutions
Ala (A) Val; Leu; Ile Val
Arg (R) Lys; Gln; Asn Lys
Asn (N) Gln; His; Asp, Lys; Arg Gln
Asp (D) Glu; Asn Glu
Cy s (C) Ser; Ala Ser
Gln (Q) Asn; Glu Asn
Glu (E) Asp; Gln Asp
Gly (G) Ala Ala
His (H) Asn; Gln; Lys; Arg Arg
Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu
Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile
Lys (K) Arg; Gln; Asn Arg
Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr
Pro (P) Ala Ala
Ser (S) Thr Thr
Thr (T) Val; Ser Ser
Trp (W) Tyr; Phe Tyr
Tyr (Y) Trp; Phe; Thr; Ser Phe
Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
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[0309] Amino acids may be grouped according to common side-chain
properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
[0310] Non-conservative substitutions will entail exchanging a member of
one of these classes
for another class.
[0311] One type of substitutional variant involves substituting one or more
hypervariable region
residues of a parent antibody (e.g. a humanized or human antibody). Generally,
the resulting
variant(s) selected for further study will have modifications (e.g.,
improvements) in certain biological
properties (e.g., increased affinity, reduced immunogenicity) relative to the
parent antibody and/or
will have substantially retained certain biological properties of the parent
antibody. An exemplary
substitutional variant is an affinity matured antibody, which may be
conveniently generated, e.g.,
using phage display-based affinity maturation techniques such as those
described herein. Briefly, one
or more HVR residues are mutated and the variant antibodies displayed on phage
and screened for a
particular biological activity (e.g. binding affinity).
[0312] Alterations (e.g., substitutions) may be made in HVRs, e.g., to
improve antibody affinity.
Such alterations may be made in HVR "hotspots," i.e., residues encoded by
codons that undergo
mutation at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods
Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the
resulting variant VH or
VL being tested for binding affinity. Affinity maturation by constructing and
reselecting from
secondary libraries has been described, e.g., in Hoogenboom et al. in Methods
in Molecular Biology
178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some
embodiments of affinity
maturation, diversity is introduced into the variable genes chosen for
maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed
mutagenesis). A
secondary library is then created. The library is then screened to identify
any antibody variants with
the desired affinity. Another method to introduce diversity involves HVR-
directed approaches, in
which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in
antigen binding may be specifically identified, e.g., using alanine scanning
mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0313] In certain embodiments, substitutions, insertions, or deletions may
occur within one or
more HVRs so long as such alterations do not substantially reduce the ability
of the antibody to bind
antigen. For example, conservative alterations (e.g., conservative
substitutions as provided herein)
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that do not substantially reduce binding affinity may be made in HVRs. Such
alterations may, for
example, be outside of antigen contacting residues in the HVRs. In certain
embodiments of the
variant VH and VL sequences provided above, each HVR either is unaltered, or
contains no more
than one, two or three amino acid substitutions.
[0314] A useful method for identification of residues or regions of an
antibody that may be
targeted for mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and
Wells (1989) Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g.,
charged residues such as arg, asp, his, lys, and glu) are identified and
replaced by a neutral or
negatively charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of
the antibody with antigen is affected. Further substitutions may be introduced
at the amino acid
locations demonstrating functional sensitivity to the initial substitutions.
Alternatively, or
additionally, a crystal structure of an antigen-antibody complex to identify
contact points between the
antibody and antigen. Such contact residues and neighboring residues may be
targeted or eliminated
as candidates for substitution. Variants may be screened to determine whether
they contain the
desired properties.
[0315] Amino acid sequence insertions include amino- and/or carboxyl-
terminal fusions ranging
in length from one residue to polypeptides containing a hundred or more
residues, as well as
intrasequence insertions of single or multiple amino acid residues. Examples
of terminal insertions
include an antibody with an N-terminal methionyl residue. Other insertional
variants of the antibody
molecule include the fusion to the N- or C-terminus of the antibody to an
enzyme (e.g. for ADEPT) or
a polypeptide which increases the serum half-life of the antibody.
b) Glycosylation variants
[0316] In certain embodiments, an anti-MerTK antibody provided herein is
altered to increase or
decrease the extent to which the antibody is glycosylated. Addition or
deletion of glycosylation sites
to an antibody may be conveniently accomplished by altering the amino acid
sequence such that one
or more glycosylation sites is created or removed.
[0317] Where the antibody comprises an Fc region, the carbohydrate attached
thereto may be
altered. Native antibodies produced by mammalian cells typically comprise a
branched, biantennary
oligosaccharide that is generally attached by an N-linkage to Asn297 of the
CH2 domain of the Fc
region. See, e.g., Wright et al. TIB TECH 15:26-32 (1997). The oligosaccharide
may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (G1cNAc), galactose, and
sialic acid, as well as a
fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide
structure. In some
embodiments, modifications of the oligosaccharide in an antibody of the
invention may be made in
order to create antibody variants with certain improved properties.
[0318] In one embodiment, antibody variants are provided having a
carbohydrate structure that
lacks fucose attached (directly or indirectly) to an Fc region. For example,
the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from
20% to 40%. The
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amount of fucose is determined by calculating the average amount of fucose
within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g.
complex, hybrid and high
mannose structures) as measured by MALDI-TOF mass spectrometry, as described
in
WO 2008/077546, for example. Asn297 refers to the asparagine residue located
at about position 297
in the Fc region (Eu numbering of Fc region residues); however, Asn297 may
also be located about
3 amino acids upstream or downstream of position 297, i.e., between positions
294 and 300, due to
minor sequence variations in antibodies. Such fucosylation variants may have
improved ADCC
function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.);
US 2004/0093621
(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to
"defucosylated" or "fucose-
deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO
2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US
2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586;
WO
2005/035778; W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol.
336:1239-1249
(2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell
lines capable of
producing defucosylated antibodies include Lec13 CHO cells deficient in
protein fucosylation (Ripka
et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 Al, Presta, L;
and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout
cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-
Ohnuki et al.
Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng.,
94(4):680-688 (2006); and
W02003/085107).
[0319] Antibody variants are further provided with bisected
oligosaccharides, e.g., in which a
biantennary oligosaccharide attached to the Fc region of the antibody is
bisected by GlcNAc. Such
antibody variants may have reduced fucosylation and/or improved ADCC function.
Examples of such
antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.);
US Patent No.
6,602,684 (Umana et al.); and US 2005/0123546 (Umana etal.). Antibody variants
with at least one
galactose residue in the oligosaccharide attached to the Fc region are also
provided. Such antibody
variants may have improved CDC function. Such antibody variants are described,
e.g., in WO
1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju,
S.).
c) Fc region variants
[0320] In certain embodiments, one or more amino acid modifications may be
introduced into
the Fc region of an anti-MerTK antibody provided herein, thereby generating an
Fc region variant.
The Fc region variant may comprise a human Fc region sequence (e.g., a human
IgGl, IgG2, IgG3 or
IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at
one or more amino
acid positions.
[0321] In certain embodiments, the invention contemplates an antibody
variant that possesses
some but not all effector functions, which make it a desirable candidate for
applications in which the
half life of the antibody in vivo is important yet certain effector functions
(such as complement and
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ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to
confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR)
binding assays can be conducted to ensure that the antibody lacks FcyR binding
(hence likely lacking
ADCC activity), but retains FcRn binding ability. The primary cells for
mediating ADCC, NK cells,
express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR
expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet,
Anna Rev. Immunol.
9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC
activity of a molecule of
interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et
al. Proc. Nat'l Acad. Sci.
USA 83:7059-7063 (1986)) and Hellstrom, Jet al., Proc. Nat'l Acad. Sci. USA
82:1499-1502 (1985);
5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-
radioactive assays methods may be employed (see, for example, ACTITm non-
radioactive cytotoxicity
assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox
96 non-
radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells
for such assays include
peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or
additionally, ADCC activity of the molecule of interest may be assessed in
vivo, e.g., in an animal
model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). Clq
binding assays may also be carried out to confirm that the antibody is unable
to bind Clq and hence
lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and
WO 2005/100402. To assess complement activation, a CDC assay may be performed
(see, for
example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg,
M.S. et al., Blood
101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743
(2004)). FcRn
binding and in vivo clearance/half life determinations can also be performed
using methods known in
the art (see, e.g., Petkova, S.B. et al., Intl Immunol. 18(12):1759-1769
(2006)).
[0322] Antibodies with reduced effector function include those with
substitution of one or more
of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No.
6,737,056). Such Fc
mutants include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270,
297 and 327, including the so-called "DANA" Fc mutant with substitution of
residues 265 and 297 to
alanine (US Patent No. 7,332,581).
[0323] Certain antibody variants with improved or diminished binding to
FcRs are described.
(See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J.
Biol. Chem. 9(2): 6591-
6604 (2001).)
[0324] In certain embodiments, an antibody variant comprises an Fc region
with one or more
amino acid substitutions which improve ADCC, e.g., substitutions at positions
298, 333, and/or 334 of
the Fc region (EU numbering of residues).
[0325] In some embodiments, alterations are made in the Fc region that
result in altered (i.e.,
either improved or diminished) Clq binding and/or Complement Dependent
Cytotoxicity (CDC), e.g.,
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as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie etal. J.
Immunol. 164: 4178-
4184 (2000).
[0326] Antibodies with increased half lives and improved binding to the
neonatal Fc receptor
(FcRn), which is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in
U52005/0014934A1
(Hinton et al.). Those antibodies comprise an Fc region with one or more
substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include those with
substitutions at one or
more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,
317, 340, 356, 360, 362,
376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue
434 (US Patent No.
7,371,826).
[0327] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No.
5,648,260; U.S.
Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region
variants.
[0328] In an exemplary embodiment, an anti-MerTK antibody disclosed herein
comprises a
LALPG mutation in the Fc region.
d) Cysteine engineered antibody variants
[0329] In certain embodiments, it may be desirable to create cysteine
engineered antibodies, e.g.,
"thioMAbs," in which one or more residues of an antibody are substituted with
cysteine residues. In
particular embodiments, the substituted residues occur at accessible sites of
the antibody. By
substituting those residues with cysteine, reactive thiol groups are thereby
positioned at accessible
sites of the antibody and may be used to conjugate the antibody to other
moieties, such as drug
moieties or linker-drug moieties, to create an immunoconjugate, as described
further herein. In
certain embodiments, any one or more of the following residues may be
substituted with cysteine:
V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy
chain; and S400 (EU
numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be
generated as
described, e.g., in U.S. Patent No. 7,521,541.
e) Antibody Derivatives
[0330] In certain embodiments, an anti-MerTK antibody provided herein may
be further
modified to contain additional nonproteinaceous moieties that are known in the
art and readily
available. The moieties suitable for derivatization of the antibody include
but are not limited to water
soluble polymers. Non-limiting examples of water soluble polymers include, but
are not limited to,
polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose,
dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-
1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or
random copolymers),
and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene
glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols
(e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde
may have advantages
in manufacturing due to its stability in water. The polymer may be of any
molecular weight, and may
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be branched or unbranched. The number of polymers attached to the antibody may
vary, and if more
than one polymer are attached, they can be the same or different molecules. In
general, the number
and/or type of polymers used for derivatization can be determined based on
considerations including,
but not limited to, the particular properties or functions of the antibody to
be improved, whether the
antibody derivative will be used in a therapy under defined conditions, etc.
[0331] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may
be selectively heated by exposure to radiation are provided. In one
embodiment, the
nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad.
Sc!. USA 102: 11600-
11605 (2005)). The radiation may be of any wavelength, and includes, but is
not limited to,
wavelengths that do not harm ordinary cells, but which heat the
nonproteinaceous moiety to a
temperature at which cells proximal to the antibody-nonproteinaceous moiety
are killed.
B. Recombinant Methods and Compositions
[0332] Antibodies may be produced using recombinant methods and
compositions, e.g., as
described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic
acid encoding an anti-
MerTK antibody described herein is provided. Such nucleic acid may encode an
amino acid sequence
comprising the VL and/or an amino acid sequence comprising the VH of the
antibody (e.g., the light
and/or heavy chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression
vectors) comprising such nucleic acid are provided. In a further embodiment, a
host cell comprising
such nucleic acid is provided. In one such embodiment, a host cell comprises
(e.g., has been
transformed with): (1) a vector comprising a nucleic acid that encodes an
amino acid sequence
comprising the VL of the antibody and an amino acid sequence comprising the VH
of the antibody, or
(2) a first vector comprising a nucleic acid that encodes an amino acid
sequence comprising the VL of
the antibody and a second vector comprising a nucleic acid that encodes an
amino acid sequence
comprising the VH of the antibody. In one embodiment, the host cell is
eukaryotic, e.g. a Chinese
Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). In one
embodiment, a
method of making an anti-MerTK antibody is provided, wherein the method
comprises culturing a
host cell comprising a nucleic acid encoding the antibody, as provided above,
under conditions
suitable for expression of the antibody, and optionally recovering the
antibody from the host cell (or
host cell culture medium).
[0333] For recombinant production of an anti-MerTK antibody, nucleic acid
encoding an
antibody, e.g., as described above, is isolated and inserted into one or more
vectors for further cloning
and/or expression in a host cell. Such nucleic acid may be readily isolated
and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of binding specifically
to genes encoding the heavy and light chains of the antibody).
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[0334] Suitable host cells for cloning or expression of antibody-encoding
vectors include
prokaryotic or eukaryotic cells described herein. For example, antibodies may
be produced in
bacteria, in particular when glycosylation and Fc effector function are not
needed. For expression of
antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos.
5,648,237, 5,789,199, and
5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C.
Lo, ed., Humana
Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody
fragments in E. coli.) After
expression, the antibody may be isolated from the bacterial cell paste in a
soluble fraction and can be
further purified.
[0335] In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast are
suitable cloning or expression hosts for antibody-encoding vectors, including
fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in the
production of an antibody
with a partially or fully human glycosylation pattern. See Gerngross, Nat.
Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0336] Suitable host cells for the expression of glycosylated antibody are
also derived from
multicellular organisms (invertebrates and vertebrates). Examples of
invertebrate cells include plant
and insect cells. Numerous baculoviral strains have been identified which may
be used in conjunction
with insect cells, particularly for transfection of Spodoptera frugiperda
cells.
[0337] Plant cell cultures can also be utilized as hosts. See, e.g., US
Patent Nos. 5,959,177,
6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTm
technology for
producing antibodies in transgenic plants).
[0338] Vertebrate cells may also be used as hosts. For example, mammalian
cell lines that are
adapted to grow in suspension may be useful. Other examples of useful
mammalian host cell lines are
monkey kidney CV1 line transformed by 5V40 (COS-7); human embryonic kidney
line (293 or 293
cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby
hamster kidney cells
(BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251
(1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-
76); human cervical
carcinoma cells (BELA); canine kidney cells (MDCK; buffalo rat liver cells
(BRL 3A); human lung
cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562);
TRI cells, as
described, e.g., in Mather et al., Annals NY. Acad. Sci. 383:44-68 (1982); MRC
5 cells; and F54 cells.
Other useful mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including DHFR-
CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and
myeloma cell lines such as
YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable
for antibody
production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B.K.C. Lo, ed.,
Humana Press, Totowa, NJ), pp. 255-268 (2003).
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C. Assays
[0339] Anti-MerTK antibodies provided herein may be identified, screened
for, or characterized
for their physical/chemical properties and/or biological activities by various
assays known in the art.
[0340] In one aspect, an antibody of the invention is tested for its
antigen binding activity, e.g.,
by known methods such as ELISA, Western blot, etc.
[0341] In another aspect, competition assays may be used to identify an
antibody that competes
with one or more of the anti-MerTK antibodies disclosed herein for binding to
MerTK. In certain
embodiments, such a competing antibody binds to the same epitope (e.g., a
linear or a conformational
epitope) that is bound by one or more of the anti-MerTK antibodies disclosed
herein. Detailed
exemplary methods for mapping an epitope to which an antibody binds are
provided in Morris (1996)
"Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana
Press, Totowa, NJ).
[0342] In an exemplary competition assay, immobilized MerTK is incubated in
a solution
comprising a first labeled antibody that binds to MerTK and a second unlabeled
antibody that is being
tested for its ability to compete with the first antibody for binding to
MerTK. The second antibody
may be present in a hybridoma supernatant. As a control, immobilized MerTK is
incubated in a
solution comprising the first labeled antibody but not the second unlabeled
antibody. After incubation
under conditions permissive for binding of the first antibody to MerTK, excess
unbound antibody is
removed, and the amount of label associated with immobilized MerTK is
measured. If the amount of
label associated with immobilized MerTK is substantially reduced in the test
sample relative to the
control sample, then that indicates that the second antibody is competing with
the first antibody for
binding to MerTK. See Harlow and Lane (1988) Antibodies: A Laboratory Manual
ch.14 (Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY).
[0343] In another aspect, assays are provided for identifying anti- MerTK
antibodies thereof
having biological activity. Biological activity may include, e.g., reducing
MerTK-mediated
phagocytic activity, reducing MerTK-mediated clearance of apoptotic cells,
and/or enhancing tumor
immunogenicity of a checkpoint inhibitor. Antibodies having such biological
activity in vivo and/or
in vitro are also provided.
[0344] In certain embodiments, an antibody of the invention is tested for
such biological activity.
Examples of assays suitable for measuring such biological activity are
described further herein,
including the Exemplification section below.
D. Immunoconjugates
[0345] The invention also provides immunoconjugates comprising an anti-
MerTK antibody
herein conjugated to one or more cytotoxic agents, such as chemotherapeutic
agents or drugs, growth
inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or
animal origin, or fragments thereof), or radioactive isotopes.
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[0346] In one embodiment, an immunoconjugate is an antibody-drug conjugate
(ADC) in which
an antibody is conjugated to one or more drugs, including but not limited to a
maytansinoid (see U.S.
Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as
monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent
Nos.
5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or
derivative thereof (see U.S.
Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,
5,773,001, and
5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al.,
Cancer Res. 58:2925-
2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et
al., Current Med.
Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006);
Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl.
Acad. Sc!. USA 97:829-834
(2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002);
King et al., J. Med.
Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate;
vindesine; a taxane such
as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0347] In another embodiment, an immunoconjugate comprises an antibody as
described herein
conjugated to an enzymatically active toxin or fragment thereof, including but
not limited to
diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from
Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-
sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,
and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin, and the tricothecenes.
[0348] In another embodiment, an immunoconjugate comprises an antibody as
described herein
conjugated to a radioactive atom to form a radioconjugate. A variety of
radioactive isotopes are
available for the production of radioconjugates. Examples include At2", In',
1125, Y90, Re186, Re188,
sm153, Bi212, p32, p+ 212
and radioactive isotopes of Lu. When the radioconjugate is used for detection,
it may comprise a radioactive atom for scintigraphic studies, for example
tc99m or 1123, or a spin
label for nuclear magnetic resonance (NMR) imaging (also known as magnetic
resonance imaging,
mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-
13, nitrogen-15, oxygen-
17, gadolinium, manganese or iron.
[0349] Conjugates of an antibody and cytotoxic agent may be made using a
variety of
bifunctional protein coupling agents such as N-succinimidy1-3-(2-
pyridyldithio) propionate (SPDP),
succinimidy1-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC),
iminothiolane (IT),
bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1),
active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido
compounds (such as bis (p-
azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-
diazoniumbenzoy1)-
ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-
active fluorine
compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be
prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-
labeled 1-
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isothiocyanatobenzy1-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is
an exemplary
chelating agent for conjugation of radionucleotide to the antibody. See
W094/11026. The linker may
be a "cleavable linker" facilitating release of a cytotoxic drug in the cell.
For example, an acid-labile
linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or
disulfide-containing linker
(Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) may
be used.
[0350] The immunuoconjugates or ADCs herein expressly contemplate, but are
not limited to
such conjugates prepared with cross-linker reagents including, but not limited
to, BMPS, EMCS,
GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-STAB, sulfo-SMCC, and sulfo-SMPB, and
SVSB
(succinimidy1-(4-vinylsulfone)benzoate) which are commercially available
(e.g., from Pierce
Biotechnology, Inc., Rockford, IL., USA).
E. Methods and Compositions for Diagnostics and Detection
[0351] In certain embodiments, any of the anti-MerTK antibodies provided
herein is useful for
detecting the presence of MerTK in a biological sample. The term "detecting"
as used herein
encompasses quantitative or qualitative detection.
[0352] In one embodiment, an anti- MerTK antibody for use in a method of
diagnosis or
detection is provided. In a further aspect, a method of detecting the presence
of MerTK in a
biological sample is provided. In certain embodiments, the method comprises
contacting the
biological sample with an anti-MerTK antibody as described herein under
conditions permissive for
binding of the anti-MerTK antibody to MerTK, and detecting whether a complex
is formed between
the anti- MerTK antibody and MerTK. Such method may be an in vitro or in vivo
method. In one
embodiment, an anti- MerTK antibody is used to select subjects eligible for
therapy with an anti-
MerTK antibody, e.g. where MerTK is a biomarker for selection of patients.
[0353] In certain embodiments, labeled anti-MerTK antibodies are provided.
Labels include, but
are not limited to, labels or moieties that are detected directly (such as
fluorescent, chromophoric,
electron-dense, chemiluminescent, and radioactive labels), as well as
moieties, such as enzymes or
ligands, that are detected indirectly, e.g., through an enzymatic reaction or
molecular interaction.
Exemplary labels include, but are not limited to, the radioisotopes 32P, '4C,
125I, 3H, and 131I,
fluorophores such as rare earth chelates or fluorescein and its derivatives,
rhodamine and its
derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase
and bacterial luciferase (U.S.
Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish
peroxidase (HRP),
alkaline phosphatase, I3-galactosidase, glucoamylase, lysozyme, saccharide
oxidases, e.g., glucose
oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase,
heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen
peroxide to oxidize a
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dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin,
spin labels,
bacteriophage labels, stable free radicals, and the like.
F. Pharmaceutical Compositions and Formulations
[0354] Also provided herein are pharmaceutical compositions and
formulations comprising an
anti-MerTK antibody, and a pharmaceutically acceptable carrier.
[0355] In some embodiments, an anti-MerTK antibody described herein is in a
formulation
comprising the antibody at an amount of about 60 mg/mL, histidine acetate in a
concentration of
about 20 mM, sucrose in a concentration of about 120 mM, and polysorbate
(e.g., polysorbate 20) in a
concentration of 0.04% (w/v), and the formulation has a pH of about 5.8. In
some embodiments, the
anti-PDL1 antibody described herein is in a formulation comprising the
antibody in an amount of
about 125 mg/mL, histidine acetate in a concentration of about 20 mM, sucrose
is in a concentration
of about 240 mM, and polysorbate (e.g., polysorbate 20) in a concentration of
0.02% (w/v), and the
formulation has a pH of about 5.5.
[0356] After preparation of the anti-MerTK antibody of interest (e.g.,
techniques for producing
antibodies which can be formulated as disclosed herein are elaborated herein
and are known in the
art), the pharmaceutical formulation comprising it is prepared. In certain
embodiments, the anti-
MerTK antibody to be formulated has not been subjected to prior lyophilization
and the formulation
of interest herein is an aqueous formulation. In certain embodiments, the anti-
MerTK antibody is a
full length antibody. In one embodiment, the anti-MerTK antibody in the
formulation is an antibody
fragment, such as an F(ab')2, in which case problems that may not occur for
the full length antibody
(such as clipping of the antibody to Fab) may need to be addressed. The
therapeutically effective
amount of anti-MerTK antibody present in the formulation is determined by
taking into account the
desired dose volumes and mode(s) of administration, for example. From about 25
mg/mL to about
150 mg/mL, or from about 30 mg/mL to about 140 mg/mL, or from about 35 mg/mL
to about 130
mg/mL, or from about 40 mg/mL to about 120 mg/mL, or from about 50 mg/mL to
about 130 mg/mL,
or from about 50 mg/mL to about 125 mg/mL, or from about 50 mg/mL to about 120
mg/mL, or from
about 50 mg/mL to about 110 mg/mL, or from about 50 mg/mL to about 100 mg/mL,
or from about
50 mg/mL to about 90 mg/mL, or from about 50 mg/mL to about 80 mg/mL, or from
about 54 mg/mL
to about 66 mg/mL is an exemplary antibody concentration in the formulation.
[0357] An aqueous formulation is prepared comprising the antibody in a pH-
buffered solution. In
some embodiments, the buffer of the present disclosure has a pH in the range
from about 5.0 to about
7Ø In certain embodiments the pH is in the range from about 5.0 to about
6.5, the pH is in the range
from about 5.0 to about 6.4, in the range from about 5.0 to about 6.3, the pH
is in the range from
about 5.0 to about 6.2, the pH is in the range from about 5.0 to about 6.1,
the pH is in the range from
about 5.5 to about 6.1, the pH is in the range from about 5.0 to about 6.0,
the pH is in the range from
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about 5.0 to about 5.9, the pH is in the range from about 5.0 to about 5.8,
the pH is in the range from
about 5.1 to about 6.0, the pH is in the range from about 5.2 to about 6.0,
the pH is in the range from
about 5.3 to about 6.0, the pH is in the range from about 5.4 to about 6.0,
the pH is in the range from
about 5.5 to about 6.0, the pH is in the range from about 5.6 to about 6.0,
the pH is in the range from
about 5.7 to about 6.0, or the pH is in the range from about 5.8 to about 6Ø
In some embodiments,
the formulation has a pH of 6.0 or about 6Ø In some embodiments, the
formulation has a pH of 5.9 or
about 5.9. In some embodiments, the formulation has a pH of 5.8 or about 5.8.
In some embodiments,
the formulation has a pH of 5.7 or about 5.7. In some embodiments, the
formulation has a pH of 5.6 or
about 5.6. In some embodiments, the formulation has a pH of 5.5 or about 5.5.
In some embodiments,
the formulation has a pH of 5.4 or about 5.4. In some embodiments, the
formulation has a pH of 5.3 or
about 5.3. In some embodiments, the formulation has a pH of 5.2 or about 5.2.
Examples of buffers
that will control the pH within this range include histidine (such as L-
histidine) or sodium acetate. In
certain embodiments, the buffer contains histidine acetate or sodium acetate
in the concentration of
about 15 mM to about 25 mM. In some embodiments, the buffer contains histidine
acetate or sodium
acetate in the concentration of about 15 mM to about 25 mM, about 16 mM to
about 25 mM, about 17
mM to about 25 mM, about 18 mM to about 25 mM, about 19 mM to about 25 mM,
about 20 mM to
about 25 mM, about 21 mM to about 25 mM, about 22 mM to about 25 mM, about 15
mM, about 16
mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22
mM, about 23
mM, about 24 mM, or about 25 mM. In one embodiment, the buffer is histidine
acetate or sodium
acetate in an amount of about 20 mM, pH 5Ø In one embodiment, the buffer is
histidine acetate or
sodium acetate in an amount of about 20 mM, pH 5.1. In one embodiment, the
buffer is histidine
acetate or sodium acetate in an amount of about 20 mM, pH 5.2. In one
embodiment, the buffer is
histidine acetate or sodium acetate in an amount of about 20 mM, pH 5.3. In
one embodiment, the
buffer is histidine acetate or sodium acetate in an amount of about 20 mM, pH
5.4. In one
embodiment, the buffer is histidine acetate or sodium acetate in an amount of
about 20 mM, pH 5.5.
In one embodiment, the buffer is histidine acetate or sodium acetate in an
amount of about 20 mM,
pH 5.6. In one embodiment, the buffer is histidine acetate or sodium acetate
in an amount of about 20
mM, pH 5.7. In one embodiment, the buffer is histidine acetate or sodium
acetate in an amount of
about 20 mM, pH 5.8. In one embodiment, the buffer is histidine acetate or
sodium acetate in an
amount of about 20 mM, pH 5.9. In one embodiment, the buffer is histidine
acetate or sodium acetate
in an amount of about 20 mM, pH 6Ø In one embodiment, the buffer is
histidine acetate or sodium
acetate in an amount of about 20 mM, pH 6.1. In one embodiment, the buffer is
histidine acetate or
sodium acetate in an amount of about 20 mM, pH 6.2. In one embodiment, the
buffer is histidine
acetate or sodium acetate in an amount of about 20 mM, pH 6.3. In one
embodiment, the buffer is
histidine acetate or sodium acetate in an amount of about 25 mM, pH 5.2. In
one embodiment, the
buffer is histidine acetate or sodium acetate in an amount of about 25 mM, pH
5.3. In one
embodiment, the buffer is histidine acetate or sodium acetate in an amount of
about 25 mM, pH 5.4.
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In one embodiment, the buffer is histidine acetate or sodium acetate in an
amount of about 25 mM,
pH 5.5. In one embodiment, the buffer is histidine acetate or sodium acetate
in an amount of about 25
mM, pH 5.6. In one embodiment, the buffer is histidine acetate or sodium
acetate in an amount of
about 25 mM, pH 5.7. In one embodiment, the buffer is histidine acetate or
sodium acetate in an
amount of about 25 mM, pH 5.8. In one embodiment, the buffer is histidine
acetate or sodium acetate
in an amount of about 25 mM, pH 5.9. In one embodiment, the buffer is
histidine acetate or sodium
acetate in an amount of about 25 mM, pH 6Ø In one embodiment, the buffer is
histidine acetate or
sodium acetate in an amount of about 25 mM, pH 6.1. In one embodiment, the
buffer is histidine
acetate or sodium acetate in an amount of about 25 mM, pH 6.2. In one
embodiment, the buffer is
histidine acetate or sodium acetate in an amount of about 25 mM, pH 6.3.
[0358] In some embodiments, the formulation further comprises sucrose in an
amount of about
60 mM to about 240 mM. In some embodiments, sucrose in the formulation is
about 60 mM to about
230 mM, about 60 mM to about 220 mM, about 60 mM to about 210 mM, about 60 mM
to about 200
mM, about 60 mM to about 190 mM, about 60 mM to about 180 mM, about 60 mM to
about 170
mM, about 60 mM to about 160 mM, about 60 mM to about 150 mM, about 60 mM to
about 140
mM, about 80 mM to about 240 mM, about 90 mM to about 240 mM, about 100 mM to
about 240
mM, about 110 mM to about 240 mM, about 120 mM to about 240 mM, about 130 mM
to about 240
mM, about 140 mM to about 240 mM, about 150 mM to about 240 mM, about 160 mM
to about 240
mM, about 170 mM to about 240 mM, about 180 mM to about 240 mM, about 190 mM
to about 240
mM, about 200 mM to about 240 mM, about 80 mM to about 160 mM, about 100 mM to
about 140
mM, or about 110 mM to about 130 mM. In some embodiments, sucrose in the
formulation is about
60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM,
about 120 mM,
about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about
180 mM, about
190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, or about 240
mM.
[0359] In some embodiments, the anti-MerTK antibody concentration in the
formulation is about
40 mg/ml to about 125 mg/ml. In some embodiments, the antibody concentration
in the formulation
is about 40 mg/ml to about 120 mg/ml, about 40 mg/ml to about 110 mg/ml, about
40 mg/ml to about
100 mg/ml, about 40 mg/ml to about 90 mg/ml, about 40 mg/ml to about 80 mg/ml,
about 40 mg/ml
to about 70 mg/ml, about 50 mg/ml to about 120 mg/ml, about 60 mg/ml to about
120 mg/ml, about
70 mg/ml to about 120 mg/ml, about 80 mg/ml to about 120 mg/ml, about 90 mg/ml
to about 120
mg/ml, or about 100 mg/ml to about 120 mg/ml. In some embodiments, the anti-
MerTK antibody
concentration in the formulation is about 60 mg/ml. In some embodiments, the
anti-MerTK antibody
concentration in the formulation is about 65 mg/ml. In some embodiments, the
anti-MerTK antibody
concentration in the formulation is about 70 mg/ml. In some embodiments, the
anti-MerTK antibody
concentration in the formulation is about 75 mg/ml. In some embodiments, the
anti-MerTK antibody
concentration in the formulation is about 80 mg/ml. In some embodiments, the
anti-MerTK antibody
concentration in the formulation is about 85 mg/ml. In some embodiments, the
anti-MerTK antibody
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concentration in the formulation is about 90 mg/ml. In some embodiments, the
anti-MerTK antibody
concentration in the formulation is about 95 mg/ml. In some embodiments, the
anti-MerTK antibody
concentration in the formulation is about 100 mg/ml. In some embodiments, the
anti-MerTK antibody
concentration in the formulation is about 110 mg/ml. In some embodiments, the
anti-MerTK antibody
concentration in the formulation is about 125 mg/ml.
[0360] In some embodiments, a surfactant is added to the anti-MerTK
antibody formulation.
Exemplary surfactants include nonionic surfactants such as polysorbates (e.g.
polysorbates 20, 80 etc)
or poloxamers (e.g. poloxamer 188, etc.). The amount of surfactant added is
such that it reduces
aggregation of the formulated antibody and/or minimizes the formation of
particulates in the
formulation and/or reduces adsorption. For example, the surfactant may be
present in the formulation
in an amount from about 0.001% to about 0.5% (w/v). In some embodiments, the
surfactant (e.g.,
polysorbate 20) is from about 0.005% to about 0.2%, from about 0.005% to about
0.1%, from about
0.005% to about 0.09%, from about 0.005% to about 0.08%, from about 0.005% to
about 0.07%, from
about 0.005% to about 0.06%, from about 0.005% to about 0.05%, from about
0.005% to about
0.04%, from about 0.008% to about 0.06%, from about 0.01% to about 0.06%, from
about 0.02% to
about 0.06%, from about 0.01% to about 0.05%, or from about 0.02% to about
0.04%. In certain
embodiments, the surfactant (e.g., polysorbate 20) is present in the
formulation in an amount of
0.005% or about 0.005%. In certain embodiments, the surfactant (e.g.,
polysorbate 20) is present in
the formulation in an amount of 0.006% or about 0.006%. In certain
embodiments, the surfactant
(e.g., polysorbate 20) is present in the formulation in an amount of 0.007% or
about 0.007%. In
certain embodiments, the surfactant (e.g., polysorbate 20) is present in the
formulation in an amount
of 0.008% or about 0.008%. In certain embodiments, the surfactant (e.g.,
polysorbate 20) is present in
the formulation in an amount of 0.009% or about 0.009%. In certain
embodiments, the surfactant
(e.g., polysorbate 20) is present in the formulation in an amount of 0.01% or
about 0.01%. In certain
embodiments, the surfactant (e.g., polysorbate 20) is present in the
formulation in an amount of 0.02%
or about 0.02%. In certain embodiments, the surfactant (e.g., polysorbate 20)
is present in the
formulation in an amount of 0.03% or about 0.03%. In certain embodiments, the
surfactant (e.g.,
polysorbate 20) is present in the formulation in an amount of 0.04% or about
0.04%. In certain
embodiments, the surfactant (e.g., polysorbate 20) is present in the
formulation in an amount of 0.05%
or about 0.05%. In certain embodiments, the surfactant (e.g., polysorbate 20)
is present in the
formulation in an amount of 0.06% or about 0.06%. In certain embodiments, the
surfactant (e.g.,
polysorbate 20) is present in the formulation in an amount of 0.07% or about
0.07%. In certain
embodiments, the surfactant (e.g., polysorbate 20) is present in the
formulation in an amount of 0.08%
or about 0.08%. In certain embodiments, the surfactant (e.g., polysorbate 20)
is present in the
formulation in an amount of 0.1% or about 0.1%. In certain embodiments, the
surfactant (e.g.,
polysorbate 20) is present in the formulation in an amount of 0.2% or about
0.2%. In certain
embodiments, the surfactant (e.g., polysorbate 20) is present in the
formulation in an amount of 0.3%
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or about 0.3%. In certain embodiments, the surfactant (e.g., polysorbate 20)
is present in the
formulation in an amount of 0.4% or about 0.4%. In certain embodiments, the
surfactant (e.g.,
poly sorbate 20) is present in the formulation in an amount of 0.5% or about
0.5%.
[0361] In one embodiment, the formulation contains the above-identified
agents (e.g., antibody,
buffer, sucrose, and/or surfactant) and is essentially free of one or more
preservatives, such as benzyl
alcohol, phenol, m-cresol, chlorobutanol and benzethonium Cl. In another
embodiment, a
preservative may be included in the formulation, particularly where the
formulation is a multidose
formulation. The concentration of preservative may be in the range from about
0.1% to about 2%,
preferably from about 0.5% to about 1%. One or more other pharmaceutically
acceptable carriers,
excipients or stabilizers such as those described in Remington's
Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980) may be included in the formulation provided that they do
not adversely affect the
desired characteristics of the formulation. Acceptable carriers, excipients or
stabilizers are nontoxic to
recipients at the dosages and concentrations employed and include; additional
buffering agents; co-
solvents; anti-oxidants including ascorbic acid and methionine; chelating
agents such as EDTA; metal
complexes (e.g. Zn-protein complexes); biodegradable polymers such as
polyesters; and/or salt-
forming counterions. Exemplary pharmaceutically acceptable carriers herein
further include
insterstitial drug dispersion agents such as soluble neutral-active
hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such
as rHuPH20
(HYLENEX , Baxter International, Inc.). Certain exemplary sHASEGPs and methods
of use,
including rHuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In
one aspect, a sHASEGP is combined with one or more additional
glycosaminoglycanases such as
chondroitinases.
[0362] The formulation herein may also contain more than one protein as
necessary for the
particular indication being treated, preferably those with complementary
activities that do not
adversely affect the other protein. For example, where the antibody is anti-
MerTK, it may be
combined with another agent (e.g., a chemotherapeutic agent and/or an anti-
neoplastic agent).
[0363] Pharmaceutical compositions and formulations as described herein can
be prepared by
mixing the active ingredients (such as an antibody or a polypeptide) having
the desired degree of
purity with one or more optional pharmaceutically acceptable carriers
(Remington 's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous
solutions. Pharmaceutically acceptable carriers are generally nontoxic to
recipients at the dosages and
concentrations employed, and include, but are not limited to: buffers such as
phosphate, citrate, and
other organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride;
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum albumin,
gelatin, or immunoglobulins;
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hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as
glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides,
and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars
such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium;
metal complexes (e.g. Zn-
protein complexes); and/or non-ionic surfactants such as polyethylene glycol
(PEG). Exemplary
pharmaceutically acceptable carriers herein further include insterstitial drug
dispersion agents such as
soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example,
human soluble PH-20
hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX , Baxter International,
Inc.). Certain
exemplary sHASEGPs and methods of use, including rHuPH20, are described in US
Patent
Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is
combined with one
or more additional glycosaminoglycanases such as chondroitinases.
[0364] Exemplary lyophilized antibody formulations are described in US
Patent No. 6,267,958.
Aqueous antibody formulations include those described in US Patent No.
6,171,586 and
W02006/044908, the latter formulations including a histidine-acetate buffer.
[0365] The composition and formulation herein may also contain more than
one active
ingredients as necessary for the particular indication being treated,
preferably those with
complementary activities that do not adversely affect each other. Such active
ingredients are suitably
present in combination in amounts that are effective for the purpose intended.
[0366] Active ingredients may be entrapped in microcapsules prepared, for
example, by
coacervation techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug
delivery systems (for example, liposomes, albumin microspheres,
microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington
's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980).
[0367] Sustained-release preparations may be prepared. Suitable examples of
sustained-release
preparations include semipermeable matrices of solid hydrophobic polymers
containing the anti-
MerTK antibody, which matrices are in the form of shaped articles, e.g. films,
or microcapsules.
The formulations to be used for in vivo administration are generally sterile.
Sterility may be readily
accomplished, e.g., by filtration through sterile filtration membranes.
III. Methods of Treatment and Uses
[0368] In one aspect, the present disclosure provides a method of treating
an individual having
cancer including administering to the individual an effective amount of an
anti-MerTK antibody as
described above.
(i) Monotherapy
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[0369] In some embodiments, an anti-MerTK antibody of the present
disclosure is administered
as a monotherapy to treat an individual having cancer. As used herein,
"cancer" refers to or describes
the physiological condition in mammals that is typically characterized by
unregulated cell growth. In
certain embodiments, the cancer may be a solid cancer or a hematologic cancer.
Solid cancers are
generally characterized by tumor mass formation in specific tissues. "Tumor,"
as used herein, refers to
all neoplastic cell growth and proliferation, whether malignant or benign, and
all pre-cancerous and
cancerous cells and tissues. Non-limiting examples of solid cancers to be
treated with an anti-MerTK
antibody of the present disclosure include carcinoma, lymphoma, blastoma, and
sarcoma. More
particular examples of such cancers include, but not limited to, squamous cell
cancer (e.g., epithelial
squamous cell cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer,
adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the
peritoneum,
hepatocellular cancer, gastric or stomach cancer including gastrointestinal
cancer and gastrointestinal
stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder
cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer,
rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or
renal cancer, prostate
cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma,
penile carcinoma,
melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral
lentiginous melanomas,
nodular melanomas, as well as abnormal vascular proliferation associated with
phakomatoses, edema
(such as that associated with brain tumors), Meigs' syndrome, brain, head and
neck cancer, and
associated metastases. In certain embodiments, cancers that are amenable to
treatment by anti-MerTK
antibodies of the present disclosure include breast cancer, colorectal cancer,
rectal cancer, non-small
cell lung cancer, glioblastoma, renal cell cancer, prostate cancer, liver
cancer, pancreatic cancer, soft-
tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer,
ovarian cancer, and
mesothelioma. In some embodiments, the cancer is selected from: small cell
lung cancer,
glioblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer,
colorectal cancer (CRC),
and hepatocellular carcinoma. Yet, in some embodiments, the cancer is selected
from: non-small cell
lung cancer, colorectal cancer, glioblastoma and breast carcinoma, including
metastatic forms of those
cancers. In some embodiments, the cancer is colorectal cancer, including colon
cancer and rectal
cancer.
[0370] In contrast, hematologic cancers originate in the blood or bone
marrow. In some
embodiments, the hematologic cancer to be treated with an anti-MerTK antibody
of the present
disclosure is leukemia. Examples of leukemias include, without limitation,
chronic lymphocytic
leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia;
chronic myeloblastic
leukemia; and acute myeloblastic leukemia. In some embodiments, the
hematologic cancer to be
treated with an anti-MerTK antibody of the present disclosure is lymphoma. Non-
limiting examples
of lymphoma include T-cell lymphoma (such as adult T-cell leukemia/lymphoma;
hepatosplenic T-
cell lymphoma; peripheral T-cell lymphoma, anaplastic large cell lymphoma; and
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angioimmunoblastic T cell lymphoma), B-cell lymphoma (including low
grade/follicular non-
Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate
grade/follicular NHL;
intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade
lymphoblastic NHL;
high grade small non-cleaved cell NHL; bulky disease NHL; diffuse large B-cell
lymphoma; mantle
cell lymphoma; Burkitt lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia), Hodgkin's lymphoma, and post-transplant
lymphoproliferative disorder
(PTLD). In some embodiments, the hematologic cancer to be treated with an anti-
MerTK antibody of
the present disclosure is myeloma. In a specific embodiment, the myeloma is
plasmacytoma or
multiple myeloma. In certain embodiments, cancers that are amenable to
treatment by anti-MerTK
antibodies of the present disclosure include non-Hodgkin's lymphoma and
multiple myeloma.
[0371] In another aspect, provided herein are methods for treating or
delaying progression of
cancer in an individual comprising administering to the individual an
effective amount of an anti-
MerTK antibody as described in the present disclosure. In some embodiments,
the treatment results
in a sustained response in the individual after cessation of the treatment.
The methods described
herein may find use in treating conditions where enhanced immunogenicity is
desired such as
increasing tumor immunogenicity for the treatment of cancer. Also provided
herein are methods of
enhancing immune function in an individual having cancer comprising
administering to the individual
an effective amount of an anti-MerTK antibody as described in the present
disclosure. In some
embodiments, the cancer expresses functional STING, functional Cx43, and
functional cGAS
polypeptides. Functional proteins are proteins that are able to carry out
their regular functions in a
cell. Examples of functional proteins may include wild-type proteins, tagged
proteins, and mutated
proteins that retain or improve protein function as compared to a wild-type
protein. Protein function
can be measured by any method known to those of skill in the art, including
assaying for protein or
mRNA expression and sequencing genomic DNA or mRNA. In some embodiments, the
cancer
comprises tumor-associated macrophages that express functional STING
polypeptides. In some
embodiments, the cancer comprises tumor cells that express functional cGAS
polypeptides. In some
embodiments, the cancer comprises tumor cells that express functional Cx43
polypeptides. In some
embodiments, the cancer is colorectal cancer, including colon cancer and
rectal cancer.
[0372] Also provided herein are methods of reducing MerTK-mediated
clearance of apoptotic
cells in an individual comprising administering to the individual an effective
amount of an anti-
MerTK antibody as described in the present disclosure to reduce MerTK-mediated
clearance of
apoptotic cells. In some embodiments, the clearance of apoptotic cells is
reduced by 1-10 fold, 1-8
fold, 1-5 fold, 1-4 fold, 1-3 fold, 1-2 fold, 2-10 fold, 2-8 fold, 2-5 fold, 2-
4 fold, 2-3 fold, 3-10 fold, 3-
8 fold, 3-5 fold, 3-4 fold, or by at least about 1.1 fold, 1.2 fold, 1.3 fold,
1.4 fold, 1.5 fold, 1.6 fold, 1.7
fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold,
2.5 fold, 2.6 fold, 2.7 fold, 2.8
fold, 2.9 fold, 3.0 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold,
3.6 fold, 3.7 fold, 3.8 fold, 3.9
fold, 4.0 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold,
4.7 fold, 4.8 fold, 4.9 fold, 5.0
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fold, 5.1 fold, 5.2 fold, 5.3 fold, 5.4 fold, 5.5 fold, 5.6 fold, 5.7 fold,
5.8 fold, 5.9 fold, 6.0 fold, 6.1
fold, 6.2 fold, 6.3 fold, 6.4 fold, 6.5 fold, 6.6 fold, 6.7 fold, 6.8 fold,
6.9 fold, 7.0 fold, 7.1 fold, 7.2
fold, 7.3 fold, 7.4 fold, 7.5 fold, 7.6 fold, 7.7 fold, 7.8 fold, 7.9 fold, or
8.0 fold. Reduction of
MerTK-mediated clearance of apoptotic cells may be determined by comparing the
level of MerTK-
mediated clearance of apoptotic cells in a sample from an individual after
administration of an
effective amount of an anti-MerTK antibody or an immunoconjugate thereof to a
reference level of
MerTK-mediated clearance of apoptotic cells. In some embodiments, the
reference level is the level
of MerTK-mediated clearance of apoptotic cells a reference sample. In some
embodiments, the
reference sample is taken from the subject taken prior to administration of an
effective amount of an
anti-MerTK antibody or an immunoconjugate thereof. In some embodiments, the
sample comprises
tumor tissue or tumor cells.
[0373] In some embodiments, an anti-MerTK antibody of the present
disclosure reduces
phagocytic activity of apoptotic cells by about 10-100%, 20-100%, 30-100%, 40-
100%, 50-100%, 60-
100%, 70-100%, 75-100%, 80-100%, 85-100%, 90-100%, 95-100%, 10-95%, 20-95%, 30-
95%, 40-
95%, 50-95%, 60-95%, 70-95%, 75-95%, 80-95%, 85-95%, 90-95%, 10-90%, 20-90%,
30-90%, 40-
90%, 50-90%, 60-90%, 70-90%, 75-90%, 80-90%, 85-90%, 10-85%, 20-85%, 30-85%,
40-85%, 50-
85%, 60-85%, 70-85%, 75-85%, 80-85%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%,
60-80%, 70-
80%, 75-80%, 10-75%, 20-75%, 30-75%, 40-75%, 50-75%, 60-75%, 70-75%, 10-70%,
20-70%, 30-
70%, 40-70%, 50-70%, 60-70%, 10-65%, 20-65%, 30-65%, 40-65%, 50-65%, 60-65%,
10-60%, 20-
60%, 30-60%, 40-60%, 50-60%, 10-55%, 20-55%, 30-55%, 40-55%, 50-55%, 10-40%,
20-40%, or
30-40%, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%,
85%, 90%,
95%, 96%, 97%, 98% or 99%. In some embodiments, the anti-MerTK antibody has a
half maximal
inhibitory concentration (IC50) for reducing phagocytic activity of apoptotic
cells of about 1 pM - 50
pM, 1 pM - 100 pM, 1 pM -500 pM, 1 pM - 1 nM, 1 pM - 1.5 nM, 5 pM-SO pM, 5 pM -
100 pM, 5
pM - 500 pM, 5 pM - 1 nM, 5 pM - 1.5 nM, 10 pM - 50 pM, 10 pM - 100 pM, 10 pM -
500 pM, 10
pM - 1 nM, 10 pM - 1.5 nM, 50 pM - 100 pM, 50 pM - 500 pM, 50 pM - 1 nM, 50 pM
- 1.5 nM,
100 pM - 500 pM, 100 pM - 1 nM, or 100 pM - 1.5 nM.
[0374] In some embodiments, the individual is a human.
[0375] The anti-MerTK antibody may be administered intravenously,
intramuscularly,
subcutaneously, topically, orally, transdermally, intraperitoneally,
intraorbitally, by implantation, by
inhalation, intrathecally, intraventricularly, or intranasally. The
appropriate dosage of the anti-MerTK
antibody may be determined based on the type of disease to be treated, the
severity and course of the
disease, the clinical condition of the individual, the individual's clinical
history and response to the
treatment, and the discretion of the attending physician.
(ii) Combinations with an Additional Therapy
[0376] In some embodiments, the uses and methods may further comprise an
additional therapy
or administration of an effective amount of an additional therapeutic agent.
The additional therapy
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may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy),
chemotherapy, gene therapy,
DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow
transplantation,
nanotherapy, monoclonal antibody therapy, or a combination of the foregoing.
The additional therapy
may be in the form of adjuvant or neoadjuvant therapy. In some embodiments,
the additional therapy
is the administration of small molecule enzymatic inhibitor or anti-metastatic
agent. In some
embodiments, the additional therapy is the administration of side-effect
limiting agents (e.g., agents
intended to lessen the occurrence and/or severity of side effects of
treatment, such as anti-nausea
agents, etc.). In some embodiments, the additional therapy is radiation
therapy. In some
embodiments, the additional therapy is surgery. In some embodiments, the
additional therapy is a
combination of radiation therapy and surgery. In some embodiments, the
additional therapy is gamma
irradiation. In some embodiments, the additional therapy is therapy targeting
PI3K/AKT/mTOR
pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or
chemopreventative agent.
[0377] In some embodiments, the additional therapy is an antagonist
directed against B7-H3
(also known as CD276), e.g., a blocking antibody, MGA271, an antagonist
directed against a TGF
beta, e.g., metelimumab (also known as CAT-192), fresolimumab (also known as
GC1008), or
LY2157299, a treatment comprising adoptive transfer of a T cell (e.g., a
cytotoxic T cell or CTL)
expressing a chimeric antigen receptor (CAR), a treatment comprising adoptive
transfer of a T cell
comprising a dominant-negative TGF beta receptor, e.g, a dominant-negative TGF
beta type II
receptor, a treatment comprising a HERCREEM protocol (see, e.g.,
ClinicalTrials.gov Identifier
NCT00889954), an agonist directed against CD137 (also known as TNFRSF9, 4-1BB,
or ILA), e.g.,
an activating antibody, urelumab (also known as BMS-663513), an agonist
directed against CD40,
e.g., an activating antibody, CP-870893, an agonist directed against 0X40
(also known as CD134),
e.g., an activating antibody, administered in conjunction with a different
anti-0X40 antibody (e.g.,
Agon0X)., an agonist directed against CD27, e.g., an activating antibody, CDX-
1127, indoleamine-
2,3-dioxygenase (IDO), 1-methyl-D-tryptophan (also known as 1-D-MT), an
antibody-drug
conjugate (in some embodiments, comprising mertansine or monomethyl auristatin
E (MMAE)), an
anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599),
trastuzumab
emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYLAO,
Genentech),
DMUC5754A, an antibody-drug conjugate targeting the endothelin B receptor
(EDNBR), e.g., an
antibody directed against EDNBR conjugated with MMAE, an angiogenesis
inhibitor, an antibody
directed against a VEGF, e.g., VEGF-A, bevacizumab (also known as AVASTINO,
Genentech), an
antibody directed against angiopoietin 2 (also known as Ang2), MEDI3617, an
antineoplastic agent,
an agent targeting CSF-1R (also known as M-CSFR or CD115), anti-CSF-1R (also
known as IMC-
CS4), an interferon, for example interferon alpha or interferon gamma, Roferon-
A, GM-CSF (also
known as recombinant human granulocyte macrophage colony stimulating factor,
rhu GM-CSF,
sargramostim, or Leukine0), IL-2 (also known as aldesleukin or Proleukin0), IL-
12, an antibody
targeting CD20 (in some embodiments, the antibody targeting CD20 is
obinutuzumab (also known as
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GA101 or Gazyva0) or rituximab), an antibody targeting GITR (in some
embodiments, the antibody
targeting GITR is TRX518), in conjunction with a cancer vaccine (in some
embodiments, the cancer
vaccine is a peptide cancer vaccine, which in some embodiments is a
personalized peptide vaccine; in
some embodiments the peptide cancer vaccine is a multivalent long peptide, a
multi-peptide, a peptide
cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see,
e.g., Yamada et al., Cancer
Sci, 104:14-21, 2013)), in conjunction with an adjuvant, a TLR agonist, e.g.,
Poly-ICLC (also known
as Hiltono10), LPS, MPL, or CpG ODN, tumor necrosis factor (TNF) alpha, IL-1,
HMGB1, an IL-10
antagonist, an IL-4 antagonist, an IL-13 antagonist, an HVEM antagonist, an
ICOS agonist, e.g., by
administration of ICOS-L, or an agonistic antibody directed against ICOS, a
treatment targeting
CX3CL1, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 or
ICAM1 agonist,
a Selectin agonist, a targeted therapy, an inhibitor of B-Raf, vemurafenib
(also known as Zelboraf0,
dabrafenib (also known as Tafinlar0), erlotinib (also known as Tarceva0), an
inhibitor of a MEK,
such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2).
cobimetinib (also
known as GDC-0973 or XL-518), trametinib (also known as Mekinist0), an
inhibitor of K-Ras, an
inhibitor of c-Met, onartuzumab (also known as MetMAb), an inhibitor of Alk,
AF802 (also known
as CH5424802 or alectinib), an inhibitor of a phosphatidylinositol 3-kinase
(PI3K), BKM120,
idelalisib (also known as GS-1101 or CAL-101), perifosine (also known as KRX-
0401), an Akt,
MK2206, GSK690693, GDC-0941, an inhibitor of mTOR, sirolimus (also known as
rapamycin),
temsirolimus (also known as CCI-779 or Torise10), everolimus (also known as
RAD001),
ridaforolimus (also known as AP-23573, MK-8669, or deforolimus), OSI-027,
AZD8055, INK128,
a dual PI3K/mTOR inhibitor, XL765, GDC-0980, BEZ235 (also known as NVP-
BEZ235),
BGT226, GSK2126458, PF-04691502, or PF-05212384 (also known as PKI-587). In
some
embodiments, the additional therapeutic agent is CT- 011 (also known as
Pidilizumab or MDV9300;
CAS Registry No. 1036730-42-3; CureTech/Medivation). CT-011, also known as
hBAT or hBAT-1,
is an antibody described in W02009/101611.
(iii) Combinations with Immune Checkpoint Inhibitors
[0378] In some embodiments, the additional therapeutic agent is an immune
checkpoint
inhibitor. In certain aspects, the application provides methods for enhancing
immune function in an
individual having cancer comprising administering an effective amount of a
combination of an anti-
MerTK antibody and an immune checkpoint inhibitor. In certain embodiments, the
anti-MERTK
antibody increases the immune effect of an immune checkpoint inhibitor by
about 2 fold, 3 fold, 5
fold, 8 fold, 10 fold, 15 fold or 20 fold. In certain embodiments, the anti-
MERTK antibody increases
the immune effect of an immune checkpoint inhibitor by about 1-2 fold, 1-5
fold, 1-10 fold, 1-15 fold,
1-20 fold, 1-25 fold, 1-30 fold, 1-50 fold, 1-75 fold, 1-100 fold, 1-150 fold,
1-200 fold, 1-250 fold,
1.5-2 fold, 1.5-5 fold, 1.5-10 fold, 1.5-15 fold, 1.5-20 fold, 1.5-25 fold,
1.5-30 fold, 1.5-50 fold, 1.5-
75 fold, 1.5-100 fold, 1.5-150 fold, 1.5-200 fold, 1.5-250 fold, 2-5 fold, 2-
10 fold, 2-15 fold, 2-20
fold, 2-25 fold, 2-30 fold, 2-50 fold, 2-75 fold, 2-100 fold, 2-150 fold, 2-
200 fold, 2-250 fold, 2.5-5
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fold, 2.5-10 fold, 2.5-15 fold, 2.5-20 fold, 2.5-25 fold, 2.5-30 fold, 2.5-50
fold, 2.5-75 fold, 2.5-100
fold, 2.5-150 fold, 2.5-200 fold, 2.5-250 fold, 5-10 fold, 5-15 fold, 5-20
fold, 5-25 fold, 5-30 fold, 5-
50 fold, 5-75 fold, 5-100 fold, 5-150 fold, 5-200 fold, 5-250 fold, 10-15
fold, 10-20 fold, 10-25 fold,
10-30 fold, 10-50 fold, 10-75 fold, 10-100 fold, 10-150 fold, 10-200 fold, 10-
250 fold, 20-25 fold, 20-
30 fold, 20-50 fold, 20-75 fold, 20-100 fold, 20-150 fold, 20-200 fold, 20-250
fold, 25-30 fold, 25-50
fold, 25-75 fold, 25-100 fold, 25-150 fold, 25-200 fold, or 25-250 fold or by
at least about 1 fold, 2
fold, 5 fold, 10 fold, 15 fold 20 fold 25 fold, 30 fold, 40 fold, 50 fold 60
fold, 70 fold, 75 fold, 80 fold,
90 fold, 100 fold, 125 fold, 150 fold, 200 fold, 225 fold or 250 fold.
[0379] In some embodiments, the individual has cancer that is resistant
(has been demonstrated
to be resistant) to one or more immune checkpoint inhibitors. In some
embodiments, resistance to
immune checkpoint inhibitors includes recurrence of cancer or refractory
cancer. Recurrence may
refer to the reappearance of cancer, in the original site or a new site, after
treatment. In some
embodiments, resistance to immune checkpoint inhibitors includes progression
of the cancer during
treatment with the immune checkpoint inhibitors. In some embodiments,
resistance to immune
checkpoint inhibitors includes cancer that does not respond to treatment. The
cancer may be resistant
at the beginning of treatment or it may become resistant during treatment. In
some embodiments, the
cancer is at early stage or at late stage.
[0380] Further details regarding therapeutic immune checkpoint inhibitors
are provided below
and in, e.g., Byun etal. (2017) Nat Rev Endocrinol. 13: 195-207; La-Beck etal.
(2015)
Pharmacotherapy. 35(10): 963-976; Buchbinder etal. (2016)Am J Clin Oncol.
39(1): 98-106; Michot
etal. (2016) Ear J Cancer. 54: 139-148, and Topalian et al. (2016) Nat Rev
Cancer. 16: 275-287.
CTL44 Inhibitors
[0381] In some embodiments, the immune checkpoint inhibitor is a cytotoxic
T-lymphocyte-
associated protein 4 (CTLA4) (also known as CD152) inhibitor. In some
embodiments, the CTLA-4
inhibitor is a blocking antibody, ipilimumab (also known as MDX-010, MDX-101,
or Yervoy0),
tremelimumab (also known as ticilimumab or CP-675,206).
PD-1 Axis Binding Antagonists
[0382] In some embodiments, the immune checkpoint inhibitor is a PD-1 axis
binding
antagonist.
[0383] Provided herein are methods for treating cancer in an individual
comprising administering
to the individual an effective amount of a PD-1 axis binding antagonist and an
anti-MerTK antibody
of the present disclosure. Also provided herein are methods of enhancing
immune function or
response in an individual (e.g., an individual having cancer) comprising
administering to the
individual an effective amount of a PD-1 axis binding antagonist and an anti-
MerTK antibody of the
present disclosure.
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[0384] In such methods, the PD-1 axis binding antagonist includes a PD-1
binding antagonist, a
PDL1 binding antagonist, and/or a PDL2 binding antagonist. Alternative names
for "PD-1" include
CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and
B7-H.
Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some
embodiments, PD-1,
PDL1, and PDL2 are human PD-1, PDL1 and PDL2.
[0385] In some embodiments, the PD-1 binding antagonist is a molecule that
inhibits the binding
of PD-1 to its ligand binding partner(s). In a specific aspect the PD-1 ligand
binding partners are
PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a
molecule that inhibits
the binding of PDL1 to its binding partner(s). In a specific aspect, PDL1
binding partner(s) are PD-1
and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule
that inhibits the
binding of PDL2 to its binding partner(s). In a specific aspect, a PDL2
binding partner is PD-1. The
antagonist may be an antibody, an antigen binding fragment thereof, an
immunoadhesin, a fusion
protein, an oligopeptide or a small molecule. If the antagonist is an
antibody, in some embodiments
the antibody comprises a human constant region selected from the group
consisting of IgGl, IgG2,
IgG3 and IgG4.
A. Anti-PD-1 Antibodies
[0386] In some embodiments, the PD-1 binding antagonist is an anti-PD-1
antibody (e.g., a
human antibody, a humanized antibody, or a chimeric antibody). A variety of
anti-PD-1 antibodies
can be utilized in the methods disclosed herein. In any of the embodiments
herein, the PD-1 antibody
can bind to a human PD-1 or a variant thereof. In some embodiments the anti-PD-
1 antibody is a
monoclonal antibody. In some embodiments, the anti-PD-1 antibody is an
antibody fragment selected
from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, and (Fab')2
fragments. In some
embodiments, the anti-PD-1 antibody is a chimeric or humanized antibody. In
other embodiments,
the anti-PD-1 antibody is a human antibody.
[0387] In some embodiments, the anti-PD-1 antibody is nivolumab (CAS
Registry Number:
946414-94-4). Nivolumab (Bristol-Myers Squibb/Ono), also known as MDX-1106-04,
MDX-1106,
ONO-4538, BMS-936558, and OPDIVOO, is an anti-PD-1 antibody described in
W02006/121168.
In some embodiments, the anti-PD-1 antibody comprises a heavy chain and a
light chain sequence,
wherein:
(a) the heavy chain comprises the amino acid sequence:
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWY
DGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
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GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLGK (SEQ ID NO: 118), and
(h) the light chain comprises the amino acid sequence:
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT
GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPS
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 119).
[0388] In some embodiments, the anti-PD-1 antibody comprises the six HVR
sequences from
SEQ ID NO: 118 and SEQ ID NO: 119 (e.g., the three heavy chain HVRs from SEQ
ID NO:118 and
the three light chain HVRs from SEQ ID NO: 119). In some embodiments, the anti-
PD-1 antibody
comprises the heavy chain variable domain from SEQ ID NO: 118 and the light
chain variable
domain from SEQ ID NO: 119.
[0389] In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS
Registry Number:
1374853-91-4). Pembrolizumab (Merck), also known as MK-3475, Merck 3475,
lambrolizumab,
SCH-900475, and KEYTRUDAO, is an anti-PD-1 antibody described in
W02009/114335. In some
embodiments, the anti-PD-1 antibody comprises a heavy chain and a light chain
sequence, wherein:
(a) the heavy chain comprises the amino acid sequence:
QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGG
INPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYW
GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK
PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ
ID NO: 120), and
(h) the light chain comprises the amino acid sequence:
EIVLTQSPAT LSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES
GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 121).
[0390] In some embodiments, the anti-PD-1 antibody comprises the six HVR
sequences from
SEQ ID NO: 120 and SEQ ID NO: 121 (e.g., the three heavy chain HVRs from SEQ
ID NO: 120 and
the three light chain HVRs from SEQ ID NO:121). In some embodiments, the anti-
PD-1 antibody
comprises the heavy chain variable domain from SEQ ID NO: 120 and the light
chain variable
domain from SEQ ID NO: 121.
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[0391] In some embodiments, the anti-PD-1 antibody is MEDI-0680 (AMP-514;
AstraZeneca).
MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.
[0392] In some embodiments, the anti-PD-1 antibody is PDR001 (CAS Registry
No. 1859072-
53-9; Novartis). PDR001 is a humanized IgG4 anti-PD-1 antibody that blocks the
binding of PDL1
and PDL2 to PD-1.
[0393] In some embodiments, the anti-PD-1 antibody is REGN2810 (Regeneron).
REGN2810 is
a human anti-PD-1 antibody.
[0394] In some embodiments, the anti-PD-1 antibody is BGB-108 (BeiGene). In
some
embodiments, the anti-PD-1 antibody is BGB-A317 (BeiGene).
[0395] In some embodiments, the anti-PD-1 antibody is JS-001 (Shanghai
Junshi). JS-001 is a
humanized anti-PD-1 antibody.
[0396] In some embodiments, the anti-PD-1 antibody is STI-A1110 (Sorrento).
STI-A1110 is a
human anti-PD-1 antibody.
[0397] In some embodiments, the anti-PD-1 antibody is INCSHR-1210 (Incyte).
INCSHR-1210
is a human IgG4 anti-PD-1 antibody.
[0398] In some embodiments, the anti-PD-1 antibody is PF-06801591 (Pfizer).
[0399] In some embodiments, the anti-PD-1 antibody is TSR-042 (also known
as ANB011;
Tesaro/AnaptysBio).
[0400] In some embodiments, the anti-PD-1 antibody is AM0001 (ARMO
Biosciences).
[0401] In some embodiments, the anti-PD-1 antibody is ENUM 244C8 (Enumeral
Biomedical
Holdings). ENUM 244C8 is an anti-PD-1 antibody that inhibits PD-1 function
without blocking
binding of PDL1 to PD-1.
[0402] In some embodiments, the anti-PD-1 antibody is ENUM 388D4 (Enumeral
Biomedical
Holdings). ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits
binding of PDL1 to
PD-1.
[0403] In some embodiments, the PD-1 antibody comprises the six HVR
sequences (e.g., the
three heavy chain HVRs and the three light chain HVRs) and/or the heavy chain
variable domain and
light chain variable domain from a PD-1 antibody described in W02015/112800
(Applicant:
Regeneron), W02015/112805 (Applicant: Regeneron), W02015/112900 (Applicant:
Novartis),
U520150210769 (Assigned to Novartis), W02016/089873 (Applicant: Celgene),
W02015/035606
(Applicant: Beigene), W02015/085847 (Applicants: Shanghai Hengrui
Pharmaceutical/Jiangsu
Hengrui Medicine), W02014/206107 (Applicants: Shanghai Junshi
Biosciences/Junmeng
Biosciences), W02012/145493 (Applicant: Amplimmune), US9205148 (Assigned to
MedImmune),
W02015/119930 (Applicants: Pfizer/Merck), W02015/119923 (Applicants:
Pfizer/Merck),
W02016/032927 (Applicants: Pfizer/Merck), W02014/179664 (Applicant:
AnaptysBio),
W02016/106160 (Applicant: Enumeral), and W02014/194302 (Applicant: Sorrento).
B. Anti-PDL1 Antibodies
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[0404] In some embodiments, the PD-1 axis binding antagonist is an anti-
PDL1 antibody. A
variety of anti-PDL1 antibodies are contemplated and described herein. In any
of the embodiments
herein, the isolated anti-PDL1 antibody can bind to a human PDL1, for example
a human PDL1 as
shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1, or a variant thereof. In
some
embodiments, the anti-PDL1 antibody is capable of inhibiting binding between
PDL1 and PD-1
and/or between PDL1 and B7-1. In some embodiments, the anti-PDL1 antibody is a
monoclonal
antibody. In some embodiments, the anti-PDL1 antibody is an antibody fragment
selected from the
group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some
embodiments, the anti-
PDL1 antibody is a humanized antibody. In some embodiments, the anti-PDL1
antibody is a human
antibody. Examples of anti-PDL1 antibodies useful for the methods of the
present disclosure, and
methods for making thereof are described in PCT patent application WO
2010/077634 Al and US
Patent No. 8,217,149, which are incorporated herein by reference.
[0405] In some embodiments, the anti-PDL1 antibody is atezolizumab (CAS
Registry Number:
1422185-06-5). Atezolizumab (Genentech), also known as MPDL3280A, is an anti-
PDL1 antibody.
[0406] In some embodiments, the anti-PDL1 antibody comprises a heavy chain
variable region
and a light chain variable region, wherein:
(a) the heavy chain variable region comprises an HVR-H1, HVR-H2, and HVR-H3
sequence of GFTFSDSWIH (SEQ ID NO: 122), AWISPYGGSTYYADSVKG (SEQ ID NO: 123)
and RHWPGGFDY (SEQ ID NO: 124), respectively, and
(b) the light chain variable region comprises an HVR-L1, HVR-L2, and HVR-L3
sequence of RASQDVSTAVA (SEQ ID NO: 125), SASFLYS (SEQ ID NO: 126) and
QQYLYHPAT
(SEQ ID NO: 127), respectively.
[0407] In some embodiments, the anti-PDL1 antibody is MPDL3280A, also known
as
atezolizumab and TECENTRIQO (CAS Registry Number: 1422185-06-5). In some
embodiments,
the anti-PDL1 antibody comprises a heavy chain and a light chain sequence,
wherein:
(a) the heavy chain variable region sequence comprises the amino acid
sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ
ID NO: 128), and
(b) the light chain variable region sequence comprises the amino acid
sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF
LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:
129).
[0408] In some embodiments, the anti-PDL1 antibody comprises a heavy chain
and a light chain
sequence, wherein:
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(a) the heavy chain comprises the amino acid sequence:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPG (SEQ ID NO: 130), and
(D) the light chain comprises the amino acid sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 131).
[0409] In some embodiments, the anti-PDL1 antibody is avelumab (CAS
Registry Number:
1537032-82-8). Avelumab, also known as MSB0010718C, is a human monoclonal IgG1
anti-PDL1
antibody (Merck KGaA, Pfizer). In some embodiments, the anti-PDL1 antibody
comprises a heavy
chain and a light chain sequence, wherein:
(a) the heavy chain comprises the amino acid sequence:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYAD
TVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPG (SEQ ID NO: 132), and
(b) the light chain comprises the amino acid sequence:
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSN
RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSS
EELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQ
WKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 133).
[0410] In some embodiments, the anti-PDL1 antibody comprises the six HVR
sequences from
SEQ ID NO: 132 and SEQ ID NO: 133 (e.g., the three heavy chain HVRs from SEQ
ID NO:132 and
the three light chain HVRs from SEQ ID NO: 133). In some embodiments, the anti-
PDL1 antibody
comprises the heavy chain variable domain from SEQ ID NO: 132 and the light
chain variable
domain from SEQ ID NO: 133.
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[0411] In some embodiments, the anti-PDL1 antibody is durvalumab (CAS
Registry Number:
1428935-60-7). Durvalumab, also known as MEDI4736, is an Fc optimized human
monoclonal IgG1
kappa anti-PDL1 antibody (MedImmune, AstraZeneca) described in W02011/066389
and
US2013/034559. In some embodiments, the anti-PDL1 antibody comprises a heavy
chain and a light
chain sequence, wherein:
(a) the heavy chain comprises the amino acid sequence:
EVQLVE S GGGLVQP GGSLRL SCAA S GFTF SRYWM SWVRQAP GKGLEWVANIKQD GSEKYY
VD SVKGRFTI SRDNAKN SLYLQMN SLRAEDTAVYYCAREGGWFGEL AFDYWGQGTLVTVS
SA STKGP SVFPLAPS SKST SGGTAALGCLVKDYFPEPVTVSWNS GALT SGVHTFPAVLQS SGL
Y SLS SVVTVP S S SL GTQTYICNVNHKP SNTKVD KRVEPKSCDKTHTCPPCPAPEFEGGP SVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPASIEKTI SKAKGQPREPQVYTLPP SREEMTKNQVSLT
CLVKGFYP SDIAVEWESNGQPENNYKTTPPVLD SD GSFFLY SKLTVD KSRWQQGNVF SC SV
MHEALHNHYTQKSLSLSPG (SEQ ID NO: 134), and
(b) the light chain comprises the amino acid sequence:
EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFS
GS GS GTDFTLTI SRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAP SVFIFPP SDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQD SKD STY SL S STLTLSKADYEK
HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 135).
[0412] In some embodiments, the anti-PDL1 antibody comprises the six HVR
sequences from
SEQ ID NO:1 34 and SEQ ID NO:135 (e.g., the three heavy chain HVRs from SEQ ID
NO:134 and
the three light chain HVRs from SEQ ID NO:135). In some embodiments, the anti-
PDL1 antibody
comprises the heavy chain variable domain from SEQ ID NO: 134 and the light
chain variable
domain from SEQ ID NO: 135.
[0413] In some embodiments, the anti-PDL1 antibody is MDX-1105 (Bristol
Myers Squibb).
MDX-1105, also known as BMS-936559, is an anti-PDL1 antibody described in
W02007/005874.
[0414] In some embodiments, the anti-PDL1 antibody is LY3300054 (Eli
Lilly).
[0415] In some embodiments, the anti-PDL1 antibody is STI-A1014 (Sorrento).
STI-A1014 is a
human anti-PDL1 antibody.
[0416] In some embodiments, the anti-PDL1 antibody is KN035 (Suzhou
Alphamab). KN035 is
single-domain antibody (dAB) generated from a camel phage display library.
[0417] In some embodiments, the anti-PDL1 antibody comprises a cleavable
moiety or linker
that, when cleaved (e.g., by a protease in the tumor microenvironment),
activates an antibody antigen
binding domain to allow it to bind its antigen, e.g., by removing a non-
binding steric moiety. In some
embodiments, the anti-PDL1 antibody is CX-072 (CytomX Therapeutics).
[0418] In some embodiments, the PDL1 antibody comprises the six HVR
sequences (e.g., the
three heavy chain HVRs and the three light chain HVRs) and/or the heavy chain
variable domain and
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light chain variable domain from a PDL1 antibody described in US20160108123
(Assigned to
Novartis), W02016/000619 (Applicant: Beigene), W02012/145493 (Applicant:
Amplimmune),
US9205148 (Assigned to MedImmune), W02013/181634 (Applicant: Sorrento), and
W02016/061142 (Applicant: Novartis).
[0419] In a still further specific aspect, the PD-1 or PDL1 antibody has
reduced or minimal
effector function. In a still further specific aspect the minimal effector
function results from an
"effector-less Fc mutation" or a glycosylation mutation. In still a further
embodiment, the effector-
less Fc mutation is an N297A or D265A/N297A substitution in the constant
region. In some
embodiments, the isolated anti-PDL1 antibody is aglycosylated. Glycosylation
of antibodies is
typically either N-linked or 0-linked. N-linked refers to the attachment of
the carbohydrate moiety to
the side chain of an asparagine residue. The tripeptide sequences asparagine-X-
serine and asparagine-
X-threonine, where X is any amino acid except proline, are the recognition
sequences for enzymatic
attachment of the carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of
these tripeptide sequences in a polypeptide creates a potential glycosylation
site. 0-linked
glycosylation refers to the attachment of one of the sugars N-
aceylgalactosamine, galactose, or xylose
to a hydroxyamino acid, most commonly serine or threonine, although 5-
hydroxyproline or 5-
hydroxylysine may also be used. Removal of glycosylation sites form an
antibody is conveniently
accomplished by altering the amino acid sequence such that one of the above-
described tripeptide
sequences (for N-linked glycosylation sites) is removed. The alteration may be
made by substitution
of an asparagine, serine or threonine residue within the glycosylation site
another amino acid residue
(e.g., glycine, alanine or a conservative substitution).
[0420] In some embodiments, the anti-MERTK antibody increases the immune
effect of the anti-
PDL1 antibody about 3 fold after 20 days of combination treatment. In some
embodiments, the anti-
MERTK antibody increases the immune effect of the anti-PDL1 antibody about 10
fold after 30 days
of treatment.
C. Other PD-1 Inhibitors
[0421] In some embodiments, the PD-1 binding antagonist is an immunoadhesin
(e.g., an
immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or
PDL2 fused to a
constant region (e.g., an Fc region of an immunoglobulin sequence). In some
embodiments, the PD-1
binding antagonist is AMP-224. AMP-224 (CAS Registry No. 1422184-00-6;
GlaxoSmithKline/MedImmune), also known as B7-DCIg, is a PDL2-Fc fusion soluble
receptor
described in W02010/027827 and W02011/066342.
[0422] In some embodiments, the PD-1 binding antagonist is a peptide or
small molecule
compound. In some embodiments, the PD-1 binding antagonist is AUNP-12
(PierreFabre/Aurigene).
See, e.g., W02012/168944, W02015/036927, W02015/044900, W02015/033303,
W02013/144704,
W02013/132317, and W02011/161699.
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[0423] In some embodiments, the PDL1 binding antagonist is a small molecule
that inhibits PD-
1. In some embodiments, the PDL1 binding antagonist is a small molecule that
inhibits PDL1. In
some embodiments, the PDL1 binding antagonist is a small molecule that
inhibits PDL1 and VISTA.
In some embodiments, the PDL1 binding antagonist is CA-170 (also known as AUPM-
170). In some
embodiments, the PDL1 binding antagonist is a small molecule that inhibits
PDL1 and TIM3. In
some embodiments, the small molecule is a compound described in W02015/033301
and
W02015/033299.
Enhancement of Immune Function
[0424] In another aspect, provided herein are methods for enhancing immune
function in an
individual having cancer comprising administering an effective amount of a
combination of an anti-
MerTK antibody and an immune checkpoint inhibitor. Various aspects of immune
function that may
be enhanced by the anti-MerTK antibodies described herein and methods for
measuring such
enhancement are described below.
[0425] In some embodiments of the methods of the present disclosure, the
cancer (in some
embodiments, a sample of the patient's cancer as examined using a diagnostic
test) has elevated levels
of T cell infiltration. As used herein, T cell infiltration of a cancer may
refer to the presence of T
cells, such as tumor-infiltrating lymphocytes (TILs), within or otherwise
associated with the cancer
tissue. It is known in the art that T cell infiltration may be associated with
improved clinical outcome
in certain cancers (see, e.g., Zhang etal., N. Engl. J. Med. 348(3):203-213
(2003)).
[0426] However, T cell exhaustion is also a major immunological feature of
cancer, with many
tumor-infiltrating lymphocytes (TILs) expressing high levels of inhibitory co-
receptors and lacking
the capacity to produce effector cytokines (Wherry, E.J. Nature immunology
12: 492-499 (2011);
Rabinovich, G.A., etal., Annual review of immunology 25:267-296 (2007)). In
some embodiments
of the methods of the present disclosure, the individual has a T cell
dysfunctional disorder. In some
embodiments of the methods of the present disclosure, the T cell dysfunctional
disorder is
characterized by T cell anergy or decreased ability to secrete cytokines,
proliferate or execute
cytolytic activity. In some embodiments of the methods of the present
disclosure, the T cell
dysfunctional disorder is characterized by T cell exhaustion. In some
embodiments of the methods of
the present disclosure, the T cells are CD4+ and CD8+ T cells. In some
embodiments, the T cells are
CD4+ and/or CD8+ T cells.
[0427] In some embodiments, CD8+ T cells are characterized, e.g., by
presence of CD8b
expression (e.g., by rtPCR using e.g., Fluidigm) (Cd8b is also known as T-cell
surface glycoprotein
CD8 beta chain; CD8 antigen, alpha polypeptide p37; Accession No. is
NM_172213). In some
embodiments, CD8+ T cells are from peripheral blood. In some embodiments, CD8+
T cells are from
tumor.
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[0428] In some embodiments, Treg cells are characterized, e.g., by presence
of Fox3p expression
(e.g., by rtPCR e.g., using Fluidigm) (Foxp3 is also known as forkhead box
protein P3; scurfin;
FOXP3delta7; immunodeficiency, polyendocrinopathy, enteropathy, X-linked; the
accession no. is
NM 014009). In some embodiments, Treg are from peripheral blood. In some
embodiments, Treg
cells are from tumor.
[0429] In some embodiments, inflammatory T cells are characterized, e.g.,
by presence of Tbet
and/or CXCR3 expression (e.g., by rtPCR using, e.g., Fluidigm). In some
embodiments,
inflammatory T cells are from peripheral blood. In some embodiments,
inflammatory T cells are from
tumor.
[0430] In some embodiments of the methods of the present disclosure, CD4
and/or CD8 T cells
exhibit increased release of cytokines selected from the group consisting of
IFN- y, TNF-a and
interleukins. Cytokine release may be measured by any means known in the art,
e.g., using Western
blot, ELISA, or immunohistochemical assays to detect the presence of released
cytokines in a sample
containing CD4 and/or CD8 T cells.
[0431] In some embodiments of the methods of the present disclosure, the
CD4 and/or CD8 T
cells are effector memory T cells. In some embodiments of the methods of the
present disclosure, the
CD4 and/or CD8 effector memory T cells are characterized by having the
expression of CD44high
CD62L10w. Expression of CD44high CD62L10w may be detected by any means known
in the art, e.g., by
preparing single cell suspensions of tissue (e.g., a cancer tissue) and
performing surface staining and
flow cytometry using commercial antibodies against CD44 and CD62L. In some
embodiments of the
methods of the present disclosure, the CD4 and/or CD8 effector memory T cells
are characterized by
having expression of CXCR3 (also known as C-X-C chemokine receptor type 3; Mig
receptor; IP10
receptor; G protein-coupled receptor 9; interferon-inducible protein 10
receptor; Accession No.
NM 001504). In some embodiments, the CD4 and/or CD8 effector memory T cells
are from
peripheral blood. In some embodiments, the CD4 and/or CD8 effector memory T
cells are from
tumor.
[0432] In some embodiments of the methods of the present disclosure, Treg
function is
suppressed relative to prior to the administration of the combination. In some
embodiments, T cell
exhaustion is decreased relative to prior to the administration of the
combination.
[0433] In some embodiments, number of Treg is decreased relative to prior
to the administration
of the combination. In some embodiments, plasma interferon gamma is increased
relative to prior to
the administration of the combination. Treg number may be assessed, e.g., by
determining percentage
of CD4+Fox3p+ CD45+ cells (e.g., by FACS analysis). In some embodiments,
absolute number of
Treg, e.g., in a sample, is determined. In some embodiments, Treg are from
peripheral blood. In some
embodiments, Treg are from tumor.
[0434] In some embodiments, T cell priming, activation and/or proliferation
is increased relative
to prior to the administration of the combination. In some embodiments, the T
cells are CD4+ and/or
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CD8+ T cells. In some embodiments, T cell proliferation is detected by
determining percentage of
Ki67+ CD8+ T cells (e.g., by FACS analysis). In some embodiments, T cell
proliferation is detected
by determining percentage of Ki67+ CD4+ T cells (e.g., by FACS analysis). In
some embodiments,
the T cells are from peripheral blood. In some embodiments, the T cells are
from tumor.
Dosage and Administration
[0435] Any of the anti-MerTK antibodies described herein and any immune
checkpoint
inhibitors known in the art or described herein may be used in the methods of
the present disclosure.
[0436] In some embodiments, the combination therapy of the present
disclosure comprises
administration of an anti-MerTK antibody and an immune checkpoint inhibitor.
The anti-MerTK
antibody and the immune checkpoint inhibitor may be administered in any
suitable manner known in
the art. For example, the anti-MerTK antibody and the immune checkpoint
inhibitor may be
administered sequentially (at different times) or concurrently (at the same
time). In some
embodiments, the immune checkpoint inhibitor is in a separate composition as
the anti-MerTK
antibody. In some embodiments, the immune checkpoint inhibitor is in the same
composition as the
anti-MerTK antibody.
[0437] The anti-MerTK antibody and the immune checkpoint inhibitor may be
administered by
the same route of administration or by different routes of administration. In
some embodiments, the
immune checkpoint inhibitor is administered intravenously, intramuscularly,
subcutaneously,
topically, orally, transdermally, intraperitoneally, intraorbitally, by
implantation, by inhalation,
intrathecally, intraventricularly, or intranasally. In some embodiments, the
anti-MerTK antibody is
administered intravenously, intramuscularly, subcutaneously, topically,
orally, transdermally,
intraperitoneally, intraorbitally, by implantation, by inhalation,
intrathecally, intraventricularly, or
intranasally. An effective amount of the immune checkpoint inhibitor and the
anti-MerTK antibody
may be administered for prevention or treatment of disease. The appropriate
dosage of the anti-
MerTK antibody and/or the immune checkpoint inhibitor may be determined based
on the type of
disease to be treated, the type of the immune checkpoint inhibitor and the
anti-MerTK antibody, the
severity and course of the disease, the clinical condition of the individual,
the individual's clinical
history and response to the treatment, and the discretion of the attending
physician. In some
embodiments, combination treatment with anti-MerTK antibody and an immune
checkpoint inhibitor
(e.g., anti- PD-1 or anti-PDL1 antibody) are synergistic, whereby an
efficacious dose of an anti-
MerTK antibody in the combination is reduced relative to efficacious dose of
the anti-MerTK
antibody as a single agent.
[0438] As a general proposition, the therapeutically effective amount of
the antibody
administered to human will be in the range of about 0.01 to about 50 mg/kg of
patient body weight
whether by one or more administrations. In some embodiments, the antibody used
is about 0.01 to
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about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg,
about 0.01 to about 30
mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01
to about 15 mg/kg,
about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to
about 1 mg/kg
administered daily, for example. In some embodiments, the antibody is
administered at 15 mg/kg.
However, other dosage regimens may be useful. In one embodiment, an anti-MerTK
antibody
described herein or an anti-PDL1 antibody described herein is administered to
a human at a dose of
about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about
600 mg, about 700
mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg,
about 1300 mg or
about 1400 mg on day 1 of 21-day cycles. The dose may be administered as a
single dose or as
multiple doses (e.g., 2 or 3 doses), such as infusions. The dose of the
antibody administered in a
combination treatment may be reduced as compared to a single treatment. The
progress of this therapy
is easily monitored by conventional techniques.
(iv) Uses
[0439] In one aspect, the present disclosure provides the anti-MerTK
antibodies as described
above for use as a medicament. In some embodiments, the use is in treating
cancer. In some
embodiments, the use is in reducing MerTK-mediated clearance of apoptotic
cells. Further provided
herein are uses of the anti-MerTK antibodies as described above in the
manufacture of a medicament.
In some embodiments, the medicament is for treatment of cancer. In some
embodiments, the cancer
expresses functional cGAS¨STING cytosolic DNA sensing pathway proteins. These
proteins are part
of the cGAS¨STING signaling pathway and function in innate immunity to detect
the presence of
cytosolic DNA in order to trigger the expression of inflammatory genes.
Examples of cGAS¨STING
cytosolic DNA sensing pathway proteins include but are not limited to cGAS,
STING, TBK-1, IRF3,
p50, p60, p65, NF-KB, ISRE, IKK, and STAT6. In some embodiments, the cancer
expresses
functional STING, functional Cx43, and functional cGAS polypeptides.
Functional proteins are
proteins that are able to carry out their regular functions in a cell.
Examples of functional proteins
may include wild-type proteins, tagged proteins, and mutated proteins that
retain or improve protein
function as compared to a wild-type protein. Protein function can be measured
by any methods known
to those of skill in the art, including assaying for protein or mRNA
expression and sequencing
genomic DNA or mRNA. In some embodiments, the cancer comprises tumor-
associated
macrophages that express functional STING polypeptides. In some embodiments,
the cancer
comprises tumor cells that express functional cGAS polypeptides. In some
embodiments, the cancer
comprises tumor cells that express functional Cx43 polypeptides. In certain
embodiments, the cancer
is colon cancer. In some embodiments, the medicament is for reducing MerTK-
mediated clearance of
apoptotic cells.
[0440] In another aspect, the individual has cancer that expresses (has
been shown to express
e.g., in a diagnostic test) PDL1 biomarker. In some embodiments, the patient's
cancer expresses low
PDL1 biomarker. In some embodiments, the patient's cancer expresses high PDL1
biomarker. In
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some embodiments of any of the methods, assays and/or kits, the PDL1 biomarker
is absent from the
sample when it comprises 0% of the sample.
[0441] In some embodiments of any of the methods, assays and/or kits, the
PDL1 biomarker is
present in the sample when it comprises more than 0% of the sample. In some
embodiments, the
PDL1 biomarker is present in at least 1% of the sample. In some embodiments,
the PDL1 biomarker
is present in at least 5% of the sample. In some embodiments, the PDL1
biomarker is present in at
least 10% of the sample.
[0442] In some embodiments of any of the methods, assays and/or kits, the
PDL1 biomarker is
detected in the sample using a method selected from the group consisting of
FACS, Western blot,
ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence,
radioimmunoassay, dot
blotting, immunodetection methods, HPLC, surface plasmon resonance, optical
spectroscopy, mass
spectrometery, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq,
microarray
analysis, SAGE, MassARRAY technique, and FISH, and combinations thereof
[0443] In some embodiments of any of the methods, assays and/or kits, the
PDL1 biomarker is
detected in the sample by protein expression. In some embodiments, protein
expression is determined
by immunohistochemistry (IHC). In some embodiments, the PDL1 biomarker is
detected using an
anti-PDL1 antibody. In some embodiments, the PDL1 biomarker is detected as a
weak staining
intensity by IHC. In some embodiments, the PDL1 biomarker is detected as a
moderate staining
intensity by IHC. In some embodiments, the PDL1 biomarker is detected as a
strong staining intensity
by IHC. In some embodiments, the PDL1 biomarker is detected on tumor cells,
tumor infiltrating
immune cells, stromal cells and any combinations thereof In some embodiments,
the staining is
membrane staining, cytoplasmic staining or combinations thereof.
[0444] In some embodiments of any of the methods, assays and/or kits, the
absence of the PDL1
biomarker is detected as absent or no staining in the sample. In some
embodiments of any of the
methods, assays and/or kits, the presence of the PDL1 biomarker is detected as
any staining in the
sample.
IV. Methods of detection
[0445] In some aspects, the present disclosure provides anti-MerTK
antibodies or
immunoconjugates thereof for use in detection of MerTK protein and cells
expression MerTK protein.
[0446] In certain embodiments, the presence and/or expression level/amount
of protein in a
sample is examined using IHC and staining protocols. IHC staining of tissue
sections has been shown
to be a reliable method of determining or detecting presence of proteins in a
sample. In some
embodiments, MerTK is detected by immunohistochemistry. In some embodiments,
elevated protein
expression is determined using IHC. In one embodiment, expression level of
MerTK is determined
using a method comprising: (a) performing IHC analysis of a sample (such as a
subject cancer
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sample) with an antibody; and b) determining expression level of the protein
in the sample. In some
embodiments, IHC staining intensity is determined relative to a reference. In
some embodiments, the
reference is a reference value. In some embodiments, the reference is a
reference sample (e.g., control
cell line staining sample or tissue sample from non-cancerous patient).
[0447] IHC may be performed in combination with additional techniques such
as morphological
staining and/or fluorescence in-situ hybridization. Two general methods of IHC
are available; direct
and indirect assays. According to the first assay, binding of antibody to the
target antigen is
determined directly. This direct assay uses a labeled reagent, such as a
fluorescent tag or an enzyme-
labeled primary antibody, which can be visualized without further antibody
interaction. In a typical
indirect assay, unconjugated primary antibody binds to the antigen and then a
labeled secondary
antibody binds to the primary antibody. Where the secondary antibody is
conjugated to an enzymatic
label, a chromogenic or fluorogenic substrate is added to provide
visualization of the antigen. Signal
amplification occurs because several secondary antibodies may react with
different epitopes on the
primary antibody.
[0448] The primary and/or secondary antibody used for IHC typically will be
labeled with a
detectable moiety. Numerous labels are available which can be generally
grouped into the following
categories: (a) Radioisotopes, such as 35S, '4C, 125I, 3H, and 131I; (b)
colloidal gold particles; (c)
fluorescent labels including, but are not limited to, rare earth chelates
(europium chelates), Texas Red,
rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin,
phycocyanin, or
commercially available fluorophores such SPECTRUM ORANGE7 and SPECTRUM GREEN7
and/or derivatives of any one or more of the above; (d) various enzyme-
substrate labels are available
and U.S. Patent No. 4,275,149 provides a review of some of these. Examples of
enzymatic labels
include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S.
Patent No. 4,737,456),
luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as
horseradish peroxidase (HRPO), alkaline phosphatase, I3-galactosidase,
glucoamylase, lysozyme,
saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-
phosphate
dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase),
lactoperoxidase,
microperoxidase, and the like.
[0449] Examples of enzyme-substrate combinations include, for example,
horseradish peroxidase
(HRPO) with hydrogen peroxidase as a substrate; alkaline phosphatase (AP) with
para-Nitrophenyl
phosphate as chromogenic substrate; and I3-D-galactosidase (13-D-Gal) with a
chromogenic substrate
(e.g., p-nitropheny1-13-D-galactosidase) or fluorogenic substrate (e.g., 4-
methylumbellifery1-13-D-
galactosidase). For a general review of these, see U.S. Patent Nos. 4,275,149
and 4,318,980.
[0450] Specimens thus prepared may be mounted and coverslipped. Slide
evaluation is then
determined, e.g., using a microscope, and staining intensity criteria,
routinely used in the art, may be
employed. In one embodiment, it is understood that when cells and/or tissue
from a tumor is
examined using IHC, staining is generally determined or assessed in tumor cell
and/or tissue (as
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opposed to stromal or surrounding tissue that may be present in the sample).
In some embodiments, it
is understood that when cells and/or tissue from a tumor is examined using
IHC, staining includes
determining or assessing in tumor infiltrating immune cells, including
intratumoral or peritumoral
immune cells.
V. Articles of Manufacture or Kits
[0451] In another embodiment of the present disclosure, an article of
manufacture or a kit is
provided comprising an anti-MerTK antibody. In some embodiments, the article
of manufacture or
kit further comprises a package insert comprising instructions for using the
anti-MerTK antibody to
treat or delay progression of cancer in an individual or to enhance immune
function of an individual
having cancer. Any of the anti-MerTK antibodies described herein may be
included in the article of
manufacture or kits. The article of manufacture or kit may further comprise an
immune checkpoint
inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-
PDL1 antibody.
[0452] In some embodiments, the immune checkpoint inhibitor and the anti-
MerTK antibody are
in the same container or separate containers. Suitable containers include, for
example, bottles, vials,
bags and syringes. The container may be formed from a variety of materials
such as glass, plastic
(such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless
steel or hastelloy). In
some embodiments, the container holds the formulation and the label on, or
associated with, the
container may indicate directions for use. The article of manufacture or kit
may further include other
materials desirable from a commercial and user standpoint, including other
buffers, diluents, filters,
needles, syringes, and package inserts with instructions for use. In some
embodiments, the article of
manufacture further includes one or more of another agent (e.g., a
chemotherapeutic agent, and anti-
neoplastic agent). Suitable containers for the one or more agent include, for
example, bottles, vials,
bags and syringes.
[0453] The specification is considered to be sufficient to enable one
skilled in the art to practice
the compositions and methods of the present disclosure. Various modifications
in addition to those
shown and described herein will become apparent to those skilled in the art
from the foregoing
description and fall within the scope of the appended claims. All
publications, patents, and patent
applications cited herein are hereby incorporated by reference in their
entirety for all purposes.
EXAMPLES
[0454] The present disclosure will be more fully understood by reference to
the following
examples. They should not, however, be construed as limiting the scope of the
disclosure. It is
understood that the examples and embodiments described herein are for
illustrative purposes only and
that various modifications or changes in light thereof will be suggested to
persons skilled in the art
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and are to be included within the spirit and purview of this application and
scope of the appended
claims.
Example 1: Generating rabbit anti-MerTK monoclonal antibodies and humanization
[0455] Monoclonal antibodies against MerTK were generated in rabbits. Then,
the antibodies
were humanized, and residues that were important for stability and affinity
were identified.
Generating rabbit anti-MerTK monoclonal antibodies
[0456] New Zealand White rabbits were immunized with human and mouse MerTK.
Individual
B-cells were isolated using a modified protocol derived from published
literature (Offner et al. PLoS
ONE 9(2), 2014). Human and mouse MerTK + B cells were sorted into single wells
using direct FACS
sorting of IgG+. B-cell culture supernatants were analyzed via primary ELISA
screening for human
and mouse MerTK binding, and B-cells were lysed and stored at -80 C.
[0457] The light chain and heavy chain variable regions of MerTK specific B
cells were
amplified by PCR and cloned into expression vectors as described in the
published literature (Offner
et al. PLoS ONE 9(2), 2014). Each recombinant rabbit monoclonal antibody was
expressed in
Expi293 cells and purified with protein A. Purified anti-MerTK antibodies were
then subjected to
functional characterization, affinity determination, and epitope binning.
[0458] Residue numbers referenced for each antibody are matched to Kabat et
al., Sequences of
proteins of immunological interest, 5th Ed., Public Health Service, National
Institutes of Health,
Bethesda, MD (1991). FIGS. 1A and 1B, respectively, show the aligned sequences
of the light chain
and heavy chain variable regions for each anti-MerTK rabbit antibody. The CDR
sequences, as
defined by Kabat et al., are underlined in FIGS. 1A and 1B.
MerTK Antibody Humanization
Step 1: Generating primary humanized antibodies
[0459] Residue numbers for each antibody referenced are matched to Kabat et
al. First,
hypervariable regions of each rabbit antibody were engineered into their
closest human germline
acceptor framework to generate primary humanized antibodies, Version 1
(labeled "v1") (human
IgG1) (FIGS, 2A-20). Specifically, the rabbit antibody light chain variable
domain (VL) positions
24-34 (L1), 50-56 (L2) and 89-97 (L3) and the heavy chain variable domain (VH)
positions 26-35
(H1), 50-65 (H2) and 95-102 (H3) were retained for the CDRs from each rabbit
antibody (FIGS. 2A
21)). In the framework, rabbit residues at "Vernier" zones, which may adjust
CDR structure and fine-
tune the antigen fit (See, e.g., Foote and Winter, J. Mol. Biol. 224: 487-499
(1992)), were also
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included. FIGS. 2A-2D show the aligned sequences of each antibody after the
first step of
humanization.
Step 2: Framework polishing humanized antibodies
[0460] Each rabbit residue at the framework "Vernier" zone of the primary
humanized antibody,
Version 1, was mutated to a human residue according to its corresponding
closest human acceptor
framework.
[0461] Each humanized mutation variant was subject to BIAcore analysis to
determine the
important rabbit residues for binding and stability. Binding affinity
determinations were obtained
using Surface Plasmon Resonance (SRP) measurements from a BIAcoreTm-T200
instrument. Briefly,
each humanized mutation variant antibody was captured to achieve approximately
100 RU (Response
Units). Then, 3-fold serial dilutions of human MerTK (0.4nM to 100nM) diluted
in HBS-EP buffer
(0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA and 0.05% v/v surfactant P20) was
injected into
the BIAcoreTm-T200 instrument at 37 C with a flow rate of 30 1/min.
Association rates (koo) and
dissociation rates (koff) were calculated using a simple one-to-one Langmuir
binding model (BIAcore
T200 evaluation software version 2.0). The equilibrium dissociation constant
(KD) was calculated as
the ratio koff/kon.
[0462] TABLES 2-5 identify the important residues for binding and stability
in gray shading.
The important residues of clone h10C3.V1 were Q2 and L4 in the light chain
variable region and 148,
G495, and K71 in the heavy chain variable region (TABLE 2). The important
residues of clone
hl OF7.V1 were L4 and F87 in the light chain variable region and V24, 148,
G49, K71, and S73 in the
heavy chain variable region (TABLE 3). The important residues of clone
h9E3.FN.V1 were L4 and
P43 in the light chain variable region and K71 in the heavy chain variable
region (TABLE 4). The
important residues of clone h13B4.V1 were G49 and V78 in the heavy chain
variable region (TABLE
5).
TABLE 2
KB (nM)
h10C3.V1 (Parent)
13.8
Q2T 17.1
LC L4M 12.3
HC Q2V 12.5
ii======== ''''''
J-IC
==============:=:=:. G49S
30.2
FTC K71R 70.0
HC S73N 15.0
HC V78L 7.4
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HC F91Y 14.9
TABLE 3
KB (nM)
h10F7.V1 (Parent)
7.3
LC A21 5.9
,....... ........ ............:.
LC F87Y 8.8
HC Q2V 5.9
FTC V24A
HC 148V 9.3
HC G49S 9.5
HC K71R 15.3
HC S731\11 7.6
HC V78L 5.1
HC F91Y 6.4
TABLE 4
KB (nM)
h9E3.FN.V1 (Parent)
12.2
LC A21 10.7
LC P43A 15.(1
HC Q2V 10.2
HC V24A 8.7
HC I48V 10.2
HC G49S 5.6
tfr-- FCi*13r:1
HC S73N 12.3
HC M78L 12.5
HC F91Y 11.5
TABLE 5
h13B4.V1 (Parent) KB (nM)
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4.8
LC V2I 5.6
LC P43A 4.6
HC Q2V 4.5
HC V24A 6.0
HC W47Y 3.1
HC I48V 5.8
HC S73N 4.7
RC. n 'V781: ...... gsZ
. ...;..
HC F91Y 4.5
HC P105R 4.2
[0463] To generate the final humanized framework polished antibodies, the
important binding
and stability rabbit framework residues were maintained while the other
residues were changed to the
closest human germline framework residues. FIGS. 2A-21) show the aligned
sequences of each
antibody including the sequences of the final humanized framework polished
antibody versions (v.14
or v.16).
[0464] A summary of the rabbit and humanized antibody sequences is provided
in TABLES 6-8.
TABLE 6
iAiifibiiitiat.tiidiiitftf4Oke1W10Wj]OWOCMMMMMMMMMIMIMMMMMMMMMMMMMM
N..4ii*M .coxttnnetiitt.2MeriAliOMMMV)).WiflCORS XEM.MOMEM
QSSPNIYS GASTLA AGGYSDSSE
MS (SEQ VISSTGGTNY VDFLVYLGGA
Rbt8F4 NYLS (SEQ S (SEQ ID AYA (SEQ ID
ID NO: 4) ASWAKG YIIWGLDL
ID NO: 1) NO: 2) NO: 3) (SEQ ID NO: 5) (SEQ ID NO: 6)
R 9E3 QSSKS SEQ SNAMS IYN
DASDLA AGGYSGDSD IISSSGSTYSAS VGFFVGYGAY
bt. NNWLS (
F S (SEQ ID YA (SEQ ID
ID NO: 10) WAKG (SEQ DYGIIHRLDL
N (SEQ ID
NO: 8) NO: 9) ID NO: 11) (SEQ ID NO: 12)
h9E3.FN NNWLS SNAMS SEQ
QSSKSIYN
DASDLA AGGYSGDSD IISSSGSTYSAS VGFFVGYGAY
(
1
S (SEQ ID YA (SEQ ID
ID NO: 10) WAKG (SEQ DYGIIHRLDL
.v (SEQ ID
NO 7) NO: 8) NO: 9) ID NO: 11) (SEQ ID NO: 12)
:
h9E3.FN NNWLS SNAMS SEQ
QSSKSIYN
DASDLA AGGYSGDSD IISSSGSTYSAS VGFFVGYGAY
(
16
S (SEQ ID YA (SEQ ID
ID NO: 10) WAKG (SEQ DYGIIHRLDL
NO 7)
.v (SEQ ID
NO: 8) NO: 9) ID NO: 11) (SEQ ID NO: 12)
:
QSSESVYN VISSGGTTYY
NDYLA Rbt10C3 SASTLAS AGGYLGNNV
GYTMG (SEQ TNWAKG VAFTAYGGGG
(SEQ ID (SEQ ID NO:
ID NO: 16) (SEQ ID NO: FPTLHRLDL
(SEQ ID
NO: 14) 15) (SEQ ID NO: 18)
NO: 13) 17)
QSSESVYN VISSGGTTYY
h10C3.v NDYLA SASTLAS AGGYLGNNV
(SEQ ID
GYTMG (SEQ TNWAKG VAFTAYGGGG
1 (SEQ ID (SEQ ID NO:
ID NO: 16) (SEQ ID NO: FPTLHRLDL
NO: 14) 15) (SEQ ID NO: 18)
NO: 13) 17)
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QSSESVYN VISSGGTTYY
SASTLAS AGGYLGNNV VAFTAYGGGG
hl0C3.v NDYLA GYTMG (SEQ TNWAKG
(SEQ ID (SEQ ID NO: FPTLHRLDL
14 (SEQ ID ID NO: 16) (SEQ ID NO:
NO: 14) 15) (SEQ ID NO: 18)
NO: 13) 17)
QSSKSVY
RASTLES AGGYSSSSSA GYAMS SEQ
VISSSGSSYYP VQFYVGYAVY
NNNWL S (
Rbt10F7 (SEQ ID (SEQ ID NA (SEQ ID ID NO 22) SWAKG (SEQ
GYGIIDRLDL
:
NO: 19) NO: 20) NO: 21) ID NO: 23) (SEQ ID NO: 24)
QSSKSVY
h1 0F7 GYAMS SEQ
RASTLES AGGYSSSSSA VISSSGSSYYP VQFYVGYAVY
.v NNNWL S (
1 SW ID
(SEQ ID NA (SEQ ID ID N 22) SWAKG (SEQ GYGIIDRLDL
(O:
NO: 19) NO: 20) NO: 21) ID NO: 23) (SEQ ID NO: 24)
QSSKSVY
h1 0F7 YAMS(SEQ RASTLES AGGYSSSSSA
VISSSGSSYYP VQFYVGYAVY
NNNWL S G : .v
(SEQ ID NA (SEQ ID SWAKG (SEQ GYGIIDRLDL
16 (SEQ ID ID NO 22)
NO: 19) NO: 20) NO: 21) ID NO: 23) (SEQ ID NO: 24)
QASQSVY
VISASGTTYY
DSKWLA SASTLAS AGAYTDNIV SW AS G AAFTAYNRGSC
SYSMG (WVN
Rbt13D8 (SEQ ID (SEQ ID (SEQ ID NO:
VIHRLDL (SEQ
28)
ID NO: 27) (SEQ ID NO:
NO: 25) NO: 14) 26) ID NO: 29)
QSSPSVYN IVSVAIDPVY
EASKLA AGGFSSGSDS SW VAFSTNGIPHR
HNWLS TYSMS ( ATWARG
Rbt22C4 S (SEQ ID FA (SEQ ID LDL (SEQ ID
SE' ID ID NO: 33) (SEI: ID NO:
NO: 31) NO: 32) NO: 35)
NO: 30) 34)
itiikiiiiiiaiiii*tikilifiiiiiiiiiiiMMMi:i:i:i:i:i:i:i:i:i:i:i:i:iiiiiiiiiiiiiii
iiiiviliii!i!i!i!iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
iiiii:i:i:i:i:i:i:i:i:i:i:i:i:i:iiiiiiiiiiiiiiiiiiiiiiiiiii:iiiiiiiiiiiiiiiiiii
iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiio
Niiien VPR LI CUR L2 ØttitIc.O.ttittilnininittiittlit CDR LL
R QASESISS SASTLAS QTYYGGSTT YGI EQ YIYPGFGITNY DLDYTGGVVG
1btl1G1 SS (S
RLA (SEQ (SEQ ID GWYV (SEQ AHSVKG (SEQ YAYVTYYFTL
ID 38)
ID NO: 36) NO: 14) ID NO: 37) NO: ID NO: 39) (SEQ ID NO: 40)
FINNVGNTYY
AS SIGN GGGGDWGYFN
AASNLA QTYYAINRY VYGMG (SEQ ASWAKG
Rbtl2H4 ALA (SEQ I (SEQ ID NO:
S (SEQ ID GGA (SEQ ID ID NO: 44) (SEQ ID NO:
ID NO: 41) 46)
NO: 42) NO: 43) 45)
IINSYGNTYY
Q ASQNIYS
R btl3B4 LA EQ
GASKLA QATYYSSNS SYAMG (SEQ ANWAKG DPGVSSNL
G (S
ID NO 47) S (SEQ ID VA (SEQ ID ID NO: 50) (SEQ ID NO:
(SEQ ID NO: 52)
:
NO: 48) NO: 49) 51)
A NIYS IINSYGNTYY
QSQ
h13B4.v LA EQ
GASKLA QATYYSSNS SYAMG (SEQ ANWAKG DPGVSSNL
G (S
1 S (SEQ ID VA (SEQ ID ID NO: 50) (SEQ ID NO:
(SEQ ID NO: 52)
ID NO: 47)
NO: 48) NO: 49) 51)
A NIYS IINSYGNTYY
QSQ
h13B4.v LA EQ
GASKLA QATYYSSNS SYAMG (SEQ ANWAKG DPGVSSNL
G (S
16 ID N S (SEQ ID VA (SEQ ID ID NO: 50) (SEQ ID NO:
(SEQ ID NO: 52)
O: 47)
NO: 48) NO: 49) 51)
IFTATGSTYY
Q ASQSISS
Rbt14C9 SLA (SE AASILAS QCTSYGSLFL ANTMN (SEQ ATWVNG SGSGSSSGAFNI
ID N 53) Q
(SEQ ID GP (SEQ ID ID NO: 56) (SEQ ID NO: (SEQ
ID NO: 58)
O:
NO: 54) NO: 55) 57)
QASQSISN AASHLA QSYFYSSTSI YAL EQ IISSTGTTYYA GAYAGYVAFG
SG (S
Rbt18G7 FLA (SEQ S (SEQ ID YNA (SEQ ID ID N TWAKG (SEQ PYYFHI (SEQ ID
62)
ID NO: 59) NO: 60) NO: 61) O: ID NO: 63) NO: 64)
TABLE 7
iiifigiiiaiiiiiiigiiiiiialitiiiiinigigigigigiMMIMMOMMIMMOMINIMI1111111111111111
1111111111111111111111111111111111111111111111111111111111111111111111111111111
11111113i=
:i:i......;:i:i:i:i:i:i:iiiiiiiiiiiii:i:i:i:i:i:i*K:
.....:..i....m_....:iii:::i:i:i:i:i.....miiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
iiiiiiiiiiiiiiiiiii:i:i:i:i:iiiiii
_.,,,...,,:::::iiiiiiiiiiiiiiimiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
iiiiiiiiiiiiiiiiiiiiiii:
orAiite. :i:iiagotit.mant YArtaivielinto.
*ilate.A.V.yivnawYAMPAO:inggfOfti:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i
:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i*:
AAVLTQTPSPVSAAVGGTVTINCQSSPNIYS QSVQESGGRLVTPGTPLTLTCTVSGFSLINYPM
NYLSWFQQKPGQPPKILIYGASTLASGVPS SWVRQAPGKGLEWIGVISSTGGTNYASWAKG
Rbt8F4 RFKGSGSGTQFTLTISDVQCDDAATYYCAG RFTISKTSTTVDLKITSPTTEDTATYFCARVDFL
GYSDSSEAYAFGGGTEVVVK (SEQ ID NO: VYLGGAYIIWGLDLWGQGTLVTVSS (SEQ ID
65) NO: 83)
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AAVLTQTP SPVSAAVGGTVSISCQS SKSIYN QSVEESGGRLVTPGTPLTLTCTVSGFSL SSNAM
NNWL SWYQQKPGQPPKLLIYDASDLA SGV SWVRQAPGKGLEWIGIIS S SG STY SA SWAKGRF
Rbt9E3.FN P SRFEGS GS GTEFTLTISDLECDDAATYY CA
TISKTSTTMDLKITSPTTEDTATYFCARVGFFVG
GGYSGDSDYAFGGGTEVVVK (SEQ ID NO: YGAYDYGIIHRLDLWGQGTLVTVSS (SEQ ID
66) NO: 84)
DAQLTQ SP STL SA S VGDRVTIT CQ SSKSIYN EQQLVESGGGLIQPGGSLRL SCAVSGF SL S SNA
NNWL SWYQQKPGKPPKLLIYDASDLA SGV MSWVRQAPGKGLEWIGIISS S GS TY SA SWAKG
h9E3 .FN.v1 P SRF S GS GS GTEFTL TIS SLQPDDFATYYCA RFTISKDS SKNTMYLQMN
SLRAEDTAVYFC AR
GGYSGDSDYAFGGGTKVEIK (SEQ ID NO: VGFFVGYGAYDYGIIHRLDLWGQGTLVTVSS
67) (SEQ ID NO: 85)
DIQLTQ SP STL SA SVGDRVTITCQ SSK SIYN EVQLVESGGGLIQPGGSLRL S CAA S GF SL S SNA
NNWL SWYQQKPGKPPKLLIYDASDLA SGV MSWVRQAPGKGLEWVSIISS S GS TY SA SWAKG
h9E3.FN.v16 P SRF S GS GS GTEFTL TIS SLQPDDFATYY CA RFTI SKDN SKNTLYL
QMNSLRAEDTAVYY CAR
GGYSGDSDYAFGGGTKVEIK (SEQ ID NO: VGFFVGYGAYDYGIIHRLDLWGQGTLVTVSS
68) (SEQ ID NO: 86)
AQVLIQTA SSVSAAVGGTVTISCQS SE S VY Q SLEES GGRL VTPGTPL TL TC TA SGF SL SGYTM
NNDYL AWYQQKP GQPPKLL IY SA S TL A S G GWVRQAPGKGLEYIGVIS SGGTTYYTNWAKG
Rbt10C3 VP SRFKGS GS GTQFTLTI SDLECDDAATYY
RFTISKTSTTVDLKITSPTTEDTATYFCARVAFT
CAGGYLGNNVFGGGTEVVVK (SEQ ID NO: AYGGGGFPTLHRLDLWGQGTLVTVSS (SEQ ID
69) NO: 87)
DQVLTQSPDSLAVSL GERATINCQS SE S VY EQQLVESGGGLVQPGGSLRL S CAA S GF SL SGYT
NNDYL AWYQQKP GQPPKLL IY SA S TL A S G MGWVRQAPGKGLEYIGVIS SGGTTYYTNWAK
h10C3N1 VPDRF S G SG S GTDFTL TI S SL QAEDVAVYY GRFTISKD S SKNTVYLQMG
SLRAEDMAVYFC A
CAGGYLGNNVFGGGTKVEIK (SEQ ID NO: RVAFTAYGGGGFPTLHRLDLWGQGTLVTVSS
70) (SEQ ID NO: 88)
DQVLTQSPDSLAVSL GERATINCQS SE S VY EVQLVESGGGLVQPGGSLRL S CAA S GF SL SGYT
NNDYL AWYQQKP GQPPKLL IY SA S TL A S G MGWVRQAPGKGLEYIGVIS SGGTTYYTNWAK
h10C3.v14 VPDRF S G SG S GTDFTL TI S SL QAEDVAVYY
GRFTISKDNSKNTLYLQMGSLRAEDMAVYYC
CAGGYLGNNVFGGGTKVEIK (SEQ ID NO: ARVAFTAYGGGGFPTLHRLDLWGQGTLVTVSS
70) (SEQ ID NO: 89)
AAVLTQTPSPVSATMGGTVSISCQS SKSVY QSVEESGGRLVTPGTPLTLTCTVSGF SL SGYAM
NNNWL SWYQQKPGQPPKLLIYRASTLESG SWVRQAPGKGLEYIGVIS S SG S SYYP SWAKGRF
Rbt10F7 VP SRFKG SGS GTQFTLTI SD VHCDDAATYF TISKTSTTVDLQIT
SPTTEDTATYFCARVQFYVG
CAGGYSSSSSANAFGGGTEVVVK (SEQ ID YAVYGYGIIDRLDLWGQGTLVTVSS (SEQ ID
NO: 71) NO: 90)
DAVLTQSPDSLAVSLGERATINCQS SK SVY EQQLVESGGGLVQPGGSLRL SCAVSGF SL SGY
NNNWL SWYQQKPGQPPKLLIYRASTLESG AMSWVRQAPGKGLEYIGVIS S S GS SYYPSWAK
h10F7.v1 VPDRF S G SG S GTDFTL TI S SL QAEDVAVYF C GRFTISKD S SKNTVYLQMG
SLRAEDMAVYFC A
AGGYSSSSSANAFGGGTKVEIK (SEQ ID RVQFYVGYAVYGYGIIDRLDLWGQGTLVTVSS
NO: 72) (SEQ ID NO: 91)
DIVLTQ SPDSLAVSL GERATINCQ SSKSVYN EVQLVESGGGLVQPGGSLRL SCAVSGF SL SGY
NNWL SWYQQKP GQPPKLL TYRA S TLES GV AMSWVRQAPGKGLEYIGVIS S S GS SYYPSWAK
h10F7.v16 PDRF S GS GS GTDFTL TI S SL QAED VAVYF C A
GRFTISKDNSKNTLYLQMGSLRAEDMAVYYC
GGYSSSSSANAFGGGTKVEIK (SEQ ID NO: ARVQFYVGYAVYGYGIIDRLDLWGQGTLVTV
73) SS (SEQ ID NO: 92)
AQVLTQTA SSVSAAVGGTVTINCQASQ SV QSLEESGGRLVTPGTPLTLTCTVSGF SF S SY SM
YD SKWL AWYQQKP GQPPKLL IY SA STLA S GWVRQAP GK GPEYIGVI SA S GTTYYA SWVNGR
Rbt13D8 GVP SRFKGSGSGTQFTLTISDLECDDAATY FTISKTSTTMDLKMTSPTAADTATYFCARAAFT
YCAGAYTDNIVFGGGTEVVVK (SEQ ID AYNRGSCVIHRLDLWGQGTLVTVSS (SEQ ID
NO: 74) NO: 93)
AQVLTQTA SSVSAAVGGTVTISCQ S SP SVY
NHNWL SWYQQKP GQPPKLL IYEASKL A SG QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYSM
SWVRQAPGKGLEWLGIVSVAIDPVYATWARG
Rbt22C4 VP SRF S GS GS GTQFTLTI SD VQ CDEAATYY
RFTISRT STTVNLKITSPTTEDTATYFCVRVAF S
NO: 75) CAGGFSSGSDSFAFGGGTEVVVT (SEQ ID
TNGIPHRLDLWGQGTLVTVSS (SEQ ID NO: 94)
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DPVLTQTPSSVEAAVGGTVTIKCQASESISS QELVESGGGLVQAGESLKLSCKASGIDFSSYGI
RLAWYQQKPGQPPKLLIYSASTLASGVSSR SWVRQAPGKGLEWIAYIYPGFGITNYAHSVKG
Rbt 1 1G11 FKGSGSGTEFTLTISDLECADAATYYCQTY RFTISSDNAQNTVFLQMPSLTASDTATYFCARD
YGGSTTGWYVFGGGTEVVVK (SEQ ID NO: LDYTGGVVGYAYVTYYFTLWGPGTLVTVSS
76) (SEQ ID NO: 95)
DVVMTQTPASVEAAVGGTVTIKCQASQSIG
QSVEESGGRLVTPGTPLTVTCTVSGFSLSVYGM
NALAWYQQKPGQRPKLLIYAASNLASGVP
GWVRQAPGKGLEYIGFINNVGNTYYASWAKG
Rbtl2H4 SRFAGSGSGTQFTLTISDLECADAATYYCQ
RFTISKTSTTVDLKITSPTTEDTATYFCAKGGGG
TYYAINRYGGAFGGGTEVVVK (SEQ ID
DWGYFNIWGPGTLVTVSL (SEQ ID NO: 96)
NO: 77)
DVVMTQTPASVSEPVGGTVTIKCQASQNIY
QSVEESGGRLVTPGTPLTLTCTVSGFSL SSYAM
SGLAWYQQKPGQPPKLLIYGASKLASGVSS
GWVRQAPGKGLEWIGIINSYGNTYYANWAKG
Rbtl3B4 RFKGSGSGTEFTLTISDLECADAATYYCQA
RFTISRTSTTVDLRMPSLTTEDTATYFCARDPG
78) TYYSSNSVAFGGGTEVVVK (SEQ ID NO:
VSSNLWGPGTLVTVSS (SEQ ID NO: 97)
DVQMTQ SP STL S A S VGDRVTITC QA SQNIY EQQLVESGEGLVQPGGSLRL SCAVSGFSL S SYA
h13B4 1 SGLAWYQQKPGKPPKLLIYGASKLASGVPS MGWVRQAPGKGLEWIGIINSYGNTYYANWAK
.v
RFSGSGSGTEFTLTISSLQPDDFATYYCQAT GRFTISRDSSKNTVYLQMGSLRAEDMAVYFCA
YYSSNSVAFGGGTKVEIK (SEQ ID NO: 79) RDPGVSSNLWGPGTLVTVSS (SEQ ID NO: 98)
DIQMTQ SP STL S A S VGDRVTIT CQA S QNIY S EVQLVESGEGLVQPGGSLRL S CAA S GF SL S
SYA
h13B4 16 GLAWYQQKPGKAPKLLIYGASKLASGVPS MGWVRQAPGKGLEYVGIINSYGNTYYANWAK
.v
RFSGSGSGTEFTLTISSLQPDDFATYYCQAT GRFTISRDNSKNTVYLQMGSLRAEDMAVYYC
YYSSNSVAFGGGTKVEIK (SEQ ID NO: 80) ARDPGVSSNLWGRGTLVTVSS (SEQ ID NO: 99)
DPVLTQTPASVSEPVGGTVTIKCQASQSIS S QSVEESGGRLVTPGTPLTLTCTVSGIDL SANTM
Rb tl4C9 SLAWYQQKPGQPPKLLIYAASILASEISSRF NWVRQAPGKGLEWIGIFTATGSTYYATWVNG
KGSRSGTEFTLTISDLECADAATYYCQCTS RFTISKTSTTVDLKITSPTTEDTATYFCARSGSG
YGSLFLGPFGGGTEVVVK (SEQ ID NO: 81) SSSGAFNIWGPGTLVTVSL (SEQ ID NO: 100)
DIVMTQTPA SVEAAVGGTVTIKCQASQ SI S QSLEESGGRLVTPGTPLTLTCTVSGIDL SSYALG
NFLAWYQQKPGQPPKVLIYAASHLASGVP WVRQAPGKGLEYIGIISSTGTTYYATWAKGRF
Rbtl8G7 SRFKGSGSGTQFTLTISDLECADAATYYCQ TISKTSSTTVDLKITGPTTEDTATYFCARGAYA
SYFYSSTSIYNAFGGGTEVVVR (SEQ ID GYVAFGPYYFHIWGPGTLVTISL (SEQ ID NO:
NO: 82) 101)
TABLE 8
EQQLVESGGGLIQPGGSLRL SCAVSGFSL S S
NAMSWVRQAPGKGLEWIGIISSSGSTYSAS
WAKGRFTISKDSSKNTMYLQMNSLRAEDT
AVYFCARVGFFVGYGAYDYGIIHRLDLWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
DAQLTQ SP STL SA SVGDRVTIT CQ S SK SIYNNNWL
AALGCLVKDYFPEPVTVSWNSGALTSGVH
SWYQQKPGKPPKLLIYDASDLASGVPSRFSGSGS
h9E3.FN.v 11,PAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPP GTEFTLTISSLQPDDFATYYCAGGYSGDSDYAFG
1 Human CPAPEAAGGPSVFLFPPKPKDTLMISRTPEV GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC
LALAPG TCVVVDVSHEDPEVKFNWYVDGVEVHNA LLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKD S TY SL SSTLTL SKADYEKHKVYACEVTHQGL
KTKPREEQYNSTYRVVSVLTVLHQDWLNG
SSPVTKSFNRGEC (SEQ ID NO: 110)
KEYKCKVSNKALGAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPG (SEQ ID NO: 102)
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EVQLVESGGGLIQPGGSLRL S CAA S GF SL S S
NAMSWVRQAPGKGLEWVSIISSSGSTYSAS
WAKGRFTISKDNSKNTLYLQMNSLRAEDT
AVYYCARVGFFVGYGAYDYGIIHRLDLWG
QGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVH DIQLTQ SP S TL SA SVGDRVTITC Q S SKSIYNNNWL
I1HPAVLQSSGLYSLSSVVTVPSSSLGTQTYI SWYQQKPGKPPKLLIYDASDLASGVPSRFSGSGS
h9E3.FN.v GTEFTLTISSLQPDDFATYYCAGGYSGDSDYAFG
CNVNHKPSNTKVDKKVEPKSCDKTHTCPP
16 Human CPAPEAAGGPSVFLFPPKPKDTLMISRTPEV GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC
LALAPG TCVVVDVSHEDPEVKFNWYVDGVEVHNA LLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
KTKPREEQYNSTYRVVSVLTVLHQDWLNG SKD S TY SL SSTLTL SKADYEKHKVYACEVTHQGL
S
KEYKCKVSNKALGAPIEKTISKAKGQPREP SPVTKSFNRGEC (SEQ ID NO: 111)
QVYTLPPSREEMTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPG (SEQ ID NO: 103)
EQQLVESGGGLVQPGGSLRL S C AA S GF SL S
GYTMGWVRQAPGKGLEYIGVISSGGTTYY
TNWAKGRFTISKDSSKNTVYLQMGSLRAE
DMAVYFCARVAFTAYGGGGFPTLHRLDL
WGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTS DQVLTQSPDSLAVSLGERATINCQSSESVYNNDY
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ LAWYQQKPGQPPKLLIYSASTLASGVPDRFSGSG
hl0C3.v1 SGTDFTLTISSLQAEDVAVYYCAGGYLGNNVFGG
Human TYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPEAAGGPSVFLFPPKPKDTLMISRT GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LALAPG PEVTCVVVDVSHEDPEVKFNWYVDGVEV LNNFYPREAKVQWKVDNALQSGNSQESVTEQDS
HNAKTKPREEQYNSTYRVVSVLTVLHQDW KD S TY SL SSTLTL SKADYEKHKVYACEVTHQGL S
LNGKEYKCKVSNKALGAPIEKTISKAKGQP SPVTKSFNRGEC (SEQ ID NO: 112)
REPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPG (SEQ ID NO: 104)
EVQLVESGGGLVQPGGSLRL S C AA S GF SL S
GYTMGWVRQAPGKGLEYIGVISSGGTTYY
TNWAKGRFTISKDNSKNTLYLQMGSLRAE
DMAVYYCARVAFTAYGGGGFPTLHRLDL
WGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTS DQVLTQSPDSLAVSLGERATINCQSSESVYNNDY
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ LAWYQQKPGQPPKLLIYSASTLASGVPDRFSGSG
hl0C3.v14 SGTDFTLTISSLQAEDVAVYYCAGGYLGNNVFGG
Human TYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPEAAGGPSVFLFPPKPKDTLMISRT GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LALAPG PEVTCVVVDVSHEDPEVKFNWYVDGVEV LNNFYPREAKVQWKVDNALQSGNSQESVTEQDS
HNAKTKPREEQYNSTYRVVSVLTVLHQDW KD S TY SL SSTLTLSKADYEKHKVYACEVTHQGL S
LNGKEYKCKVSNKALGAPIEKTISKAKGQP SPVTKSFNRGEC (SEQ ID NO: 113)
REPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPG (SEQ ID NO: 105)
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EQQLVESGGGLVQPGGSLRL S C AV S GF SL S
GYAMSWVRQAPGKGLEYIGVISSSGSSYYP
SWAKGRFTISKDSSKNTVYLQMGSLRAED
MAVYFCARVQFYVGYAVYGYGIIDRLDL
WGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTS DAVLTQSPDSLAVSLGERATINCQSSKSVYNNNW
L SWYQQKPGQPPKWYRASTLE SGVPDRFS GS G S
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
hl0F7.v1 GTDFTLTISSLQAEDVAVYFCAGGYSSSSSANAFG
Human TYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPEAAGGPSVFLFPPKPKDTLMISRT GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC
LALAPG PEVTCVVVDVSHEDPEVKFNWYVDGVEV LLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
HNAKTKPREEQYNSTYRVVSVLTVLHQDW SKD S TY SL SSTLTL SKADYEKHKVYACEVTHQGL
S
LNGKEYKCKVSNKALGAPIEKTISKAKGQP SPVTKSFNRGEC (SEQ ID NO: 114)
REPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPG (SEQ ID NO: 106)
EVQLVESGGGLVQPGGSLRL S C AV S GF SL S
GYAMSWVRQAPGKGLEYIGVISSSGSSYYP
SWAKGRFTISKDNSKNTLYLQMGSLRAED
MAVYYCARVQFYVGYAVYGYGIIDRLDL
WGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTS DIVLTQSPDSLAVSLGERATINCQSSKSVYNNNW
L SWYQQKPGQPPKWYRASTLE SGVPDRFS GS G S
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
hl0F7.v16 GTDFTLTISSLQAEDVAVYFCAGGYSSSSSANAFG
Human TYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPEAAGGPSVFLFPPKPKDTLMISRT GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC
LALAPG PEVTCVVVDVSHEDPEVKFNWYVDGVEV LLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
HNAKTKPREEQYNSTYRVVSVLTVLHQDW SKD S TY SL SSTLTL SKADYEKHKVYACEVTHQGL
S
LNGKEYKCKVSNKALGAPIEKTISKAKGQP SPVTKSFNRGEC (SEQ ID NO: 115)
REPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPG (SEQ ID NO: 107)
EQQLVESGEGLVQPGGSLRL SCAVSGFSL S
SYAMGWVRQAPGKGLEWIGIINSYGNTYY
ANVVAKGRFTISRDSSKNTVYLQMGSLRAE
DMAVYFCARDPGVSSNLWGPGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSG DVQMTQ SP STL SA SVGDRVTITCQASQNIYS GL A
WYQQKPGKPPKWYGASKLASGVPSRFSGSGSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
h13B4.v1 KVDKKVEPKSCDKTHTCPPCPAPEAAGGPS TEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGG
Human TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
VFLFPPKPKDTLMISRTPEVTCVVVDVSHE
LALAPG DPEVKFNWYVDGVEVHNAKTKPREEQYN NNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
STYRVVSVLTVLHQDWLNGKEYKCKVSN D STY SL S STLTL SKADYEKHKVYACEVTHQGL S S
KALGAPIEKTISKAKGQPREPQVYTLPPSRE PVTKSFNRGEC (SEQ ID NO: 116)
EMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLS
PG (SEQ ID NO: 108)
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EVQLVESGEGLVQPGGSLRL SCAASGFSL S
SYAMGWVRQAPGKGLEYVGIINSYGNTYY
ANWAKGRFTISRDNSKNTVYLQMGSLRAE
DMAVYYCARDPGVSSNLWGRGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DIQMTQSPSTLSASVGDRVTITCQASQNIYSGLA
DYFPEPVTVSWNSGALTSGVHTFPAVLQSS WYQQKPGKAPKLLIYGASKLASGVPSRFSGSGSG
GLYSL S SVVTVPS SSLGTQTYICNVNHKPSN
h13B4.v16 TEFTLTISSLQPDDFATYYCQATYYSSNSVAFGGG
TKVDKKVEPKSCDKTHTCPPCPAPEAAGGP
Human TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
LALAPG NNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DPEVKFNWYVDGVEVHNAKTKPREEQYN
D STYSL S STLTL SKADYEKHKVYACEVTHQGL S S
STYRVVSVLTVLHQDWLNGKEYKCKVSN
PVTKSFNRGEC (SEQ ID NO: 117)
KALGAPIEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLS
PG (SEQ ID NO: 109)
[0465] In certain embodiments, each of SEQ ID NOs: 102-109 may optionally
comprise a lysine
(K) at the C-terminal end of the amino acid sequence, e.g., each sequence may
end in PGK rather than
in PG.
Example 2: Antibody binding affinity
[0466] Each rabbit and humanized antibody was subjected to a binding assay
to determine its
affinity to MerTK derived from various species.
[0467] All binding affinity determinations were obtained using Surface
Plasmon Resonance
(SPR) measurements from a BIAcoreTm-T200 instrument. Briefly, each rabbit or
humanized antibody
was captured to achieve approximately 100 RU (Response Units). Then, 3-fold
serial dilutions of
MerTK from various species (0.4nM to 100nM) diluted in HBS-EP buffer (0.01M
HEPES pH 7.4,
0.15M NaCl, 3mM EDTA and 0.05% v/v surfactant P20) was injected into the
BIAcoreTm-T200
instrument at 25 C or 37 C with a flow rate of 30 1/min. Association rates
(kon) and dissociation rates
(koff) were calculated using a simple one-to-one Langmuir binding model
(BIAcore T200 evaluation
software version 2.0). The equilibrium dissociation constant (KID) was
calculated as the ratio koff/kon.
[0468] TABLE 9 shows the equilibrium dissociation constant, KID, measured
via BIAcore
analysis for each rabbit anti-MerTK antibody binding to human, cynomolgus
monkey, and mouse
MerTK protein. TABLES 10-13 compare the KID measured for rabbit anti-MerTK
monoclonal
antibodies to their matched antibodies after the first step of humanization
(V1). TABLES 14-17
compare the KID for each antibody binding to human, cynomolgus monkey, rat,
and mouse MerTK
protein, after the final step of humanization (humanized polished mAb) to the
KID of the same
antibody after the first step of humanization (V1). The polished humanized mAb
are hl0C3.v14,
h9E3.FN.v1, hl 0F7.v16, and h13B4.v16 respectively.
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TABLE 9
BIAcore (ICD:nM) at 25 C
MerTK
Human Cyno Mouse
Antibody
MerTK MerTK MerTK
Rbt8F4 44 14.7 2.3
Rbt9E3.FN 2.6 2.5 0.6
Rbtl 0C3 3.4 3.0 0.7
Rbt10F7 7.0 4.7 4.1
Rbtl 1G11 31.3 14.3 4.1
Rbt12H4 18 13.9 8.5
Rbt13B4 2.9 1.8 >1000
Rbt13D8 4.3 3.8 1.2
Rbt14C9 >1000 NA 0.6
Rbt18G7 2.3 4.3 1.7
Rbt22C4 94 82.2 2.2
TABLE 10
Clone 10C3 (FN Binder) at 37 C
Human Cyno Rat Mouse
MerTK KD: KD: KD: KD:
nM nM nM nM
Rabbit mAb 9.9 4.1 2.4 0.8
Humanized
12.9 5.3 3.3 1.1
mAb V1
TABLE 11
Clone 9E3.FN (FN Binder) at 37 C
Human Cyno Rat Mouse
MerTK KD: KD: KD: KD:
nM nM nM nM
Rabbit mAb 5.1 2.5 1.7 0.6
Humanized
10.6 4.5 3.7 1.5
mAb V1
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TABLE 12
Clone 10F7 (FN Binder) at 37 C
Human Cyno Rat Mouse
MerTK KD: KD: KD: KD:
nM nM nM nM
Rabbit mAb 10.9 5.1 5.8 2.4
Humanized
7.1 3.1 3.6 1.5
mAb V1
TABLE 13
Clone 13B4 (Ig Binder) at 37 C
Human Cyno Rat Mouse
MerTK KD: KD: KD: KD:
nM nM nM nM
Rabbit mAb 4.9 5.9 >100 >100
Humanized
4.8 5.6 >100 >100
mAb V1
TABLE 14
Clone 10C3 (FN Binder) at 37 C
Human Cyno Rat Mouse
MerTK KD: KD: KD: KD:
nM nM nM nM
Humanized
11.6 5.3 3.3 1
V1 mAb
*Humanized
6.9 3.2 2.4 0.9
polished mAb
TABLE 15
Clone 9E3.FN (FN Binder) at 37 C
Human Cyno Rat Mouse
MerTK KD: KD: KD: KD:
nM nM nM nM
Humanized
11.2 4.8 3.8 1.4
V1 mAb
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*Humanized
5.8 2.5 3 1.1
polished mAb
TABLE 16
Clone 10F7 (FN Binder) at 37 C
Human Cyno Rat Mouse
MerTK KD: KD: KD: KD:
nM nM nM nM
Humanized
6.7 3 4.1 1.5
V1 mAb
*Humanized
4.9 2.1 3.2 1.4
polished mAb
TABLE 17
Clone 13B4 (Ig Binder) at 37 C
Human Cyno Rat Mouse
MerTK KD: KD: KD: KD:
nM nM nM nM
Humanized
4.3 5 >100 >100
V1 mAb
*Humanized
5.1 5.7 >100 >100
polished mAb
[0469] The results confirmed that most of the rabbit antibodies are cross
species MerTK binders,
except for 14C9, which is a mouse specific MerTK binder and 13B4, which is a
human specific
MerTK binder. The results further indicated that after step 1 humanization,
there is a slight affinity
improvement against all four species of MerTK for antibody 10F7, but not for
10C3 and 9E3.FN,
which show slight affinity drop. For antibody 13B4, it is comparable before
and after humanization.
After step 2 of humanization, affinity improved against all four species of
MerTK for 10C3, 9E3.FN,
and 10F7, but not for 13B4.
Example 3: Antibody epitope characterization
[0470] The isolated anti-MerTK antibodies were characterized by epitope
binning and binding
analysis to determine epitope domain specificity.
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Epitope Binning
[0471] A 96 x 96 array-based SPR imaging system (Carterra USA) was used to
epitope bin a
panel of MerTK monoclonal antibodies. First, each anti-MerTK rabbit antibody,
diluted at lOug/m1 in
10mM sodium acetate buffer pH4.5, was directly immobilized onto a SPR
sensorprism CMD 200M
sensor chip (XanTec Bioanaiytics, Germany) using amine-coupling chemistry in a
Continuous Flow
Microspotter (Carterra, USA). Then, MerTK, at 100nM, was injected over the
sensor chip for 4
minutes to allow binding, followed by another 4 minute binding of each binning
rabbit antibody at
lOug/ml.
[0472] The surface was regenerated between each cycle using 10mM Glycine
pH1.5, and the
experiment was conducted at 25 C using HBS-EP buffer (0.01M HEPES pH 7.4,
0.15M NaCl, 3mM
EDTA and 0.05% surfactant P20). The IBIS MX96 SPRi instrument (Carterra USA)
was used to
record the binding response to the immobilized antibodies. The binding data
was analyzed using
Wasatch binning software tool to generate an epitope network plot.
[0473] The results of the binning experiment in FIG. 3 indicate which
antibodies compete for
binding with each other on certain MerTK epitopes. Antibodies 8F4, 22C4, and
13D8, raised against
mouse MerTK, and antibodies 10C3, 9E3.FN, 10F7, 22C4, 8F4, and 13D8, raised
against human
MerTK, competed for binding with each other (FIG. 3). Antibodies 12H4, 18G7,
14C9, and 11G11,
raised against mouse MerTK, and antibodies 13B4, 12H4, 18G7, and 11G11, raised
against human
MerTK, competed with each other (FIG. 3). As described below, antibodies 10C3,
9E3.FN, 10F7,
22C4, 8F4, and 13D8 bind to MerTK's fibronectin-like domain, and antibodies
13B4, 12H4, 18G7,
and 11G11 bind to MerTK's Ig-like domain.
Epitope Binding Analysis
[0474] Epitope specificity of the rabbit antibodies was also determined by
binding experiments.
Each rabbit antibody was tested for binding to four domains from human MerTK
or mouse MerTK:
the extracellular domain (HuMER R26-A499 or MuMER E23-5496), which includes
both Ig-like
domains and both fibronectin-like domains, the Ig-like 1&2 domains (HuMER G76-
P284 or MuMER
A70-P279), the Ig-like 1 domain (HuMER G76-G195 or MuMER A70-G190), and the Ig-
like 2
domain (HuMER G195-P284 or MuMER G190-P279).
[0475] Binding affinity determinations were obtained using Surface Plasmon
Resonance (SRP)
measurements from a BIAcoreTm-T200 instrument. Briefly, each rabbit antibody
was captured to
achieve approximately 100 RU (Response Units). Then, 3-fold serial dilutions
of the various MerTK
domains (0.4nM to 100nM) diluted in HBS-EP buffer (0.01M HEPES pH 7.4, 0.15M
NaCl, 3mM
EDTA and 0.05% v/v surfactant P20) was injected into the BIAcoreTm-T200
instrument at 25 C or
37 C with a flow rate of 301.11/min. Association rates (koo) and dissociation
rates (koff) were calculated
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using a simple one-to-one Langmuir binding model (BIAcore T200 evaluation
software version 2.0).
The equilibrium dissociation constant (Ku) was calculated as the ratio
kore/kon.
[0476] Antibody epitope determination was assessed by BIAcore analysis for
rabbit antibodies
binding against both the human and mouse MerTK extracellular domain (HuMER R26-
A499 and
MuMER E23-S496), Ig1&2 domain, Igl-only domain, and Ig2-only domain (TABLE
18). Human
MerTK and its domains are shown in light gray, while mouse MerTK and its
domains are shown in
dark gray.
[0477] The results of the epitope binding analysis demonstrate that cross-
reactive FN domain
antibodies, Rbt8F4, Rbt22C4 and Rbt13D8, do not bind human or mouse Igl and
Ig2 domains at 1
uM (TABLE 18). Epitope binding data was not collected for antibodies
Rbt9E3.FN, Rbt10C3 and
Rbt10F7. However, Wasatch binning demonstrated that the epitope-specificity of
Rbt9E3.FN,
Rbt10C3 and Rbtl OF7 overlaps with FN domain antibodies, Rbt8F4, Rbt22C4 and
Rbt13D8 (FIG. 3).
Therefore, the results suggested that Rbt9E3.FN, Rbt10C3 and Rbtl OF7 are FN
binding domain
antibodies that do not bind the isolated Igl and Ig2 domains.
[0478] The results of the epitope binding analysis further demonstrate that
antibodies Rbt11G11,
Rbt12H4, Rbt18G7, Rbt13B4, and Rbt14C9, are Ig domain binding antibodies
(TABLE 18).
Antibodies Rbtl1G11, Rbt12H4, and Rbt18G7 are cross-reactive Ig domain
antibodies that bind both
human and mouse MerTK Ig (TABLE 18). In contrast, Rbt13B4 and Rbt14C9 are
species-specific Ig
domain antibodies which bind human and mouse Ig, respectively (TABLE 18).
TABLE 18
BIAeore Analysis (KD: nM) at 25 C
Epitope
MerTK HuMER.
on HuMER HuMER. HuMER. MuMER MuM,ER. u MMER. MuMER.
Antibody Ig18z2 10,0 1,9,2
HuMER (R26- 76- Igl (G76- Ig2 (G195- (E23- Igl (A70-
(G (A70- 1C191)-
A499) G195) P284) -
P284) S496) C190) :::i:::
P279) ::: ::=.: P279)
..
FN Rbt8F4 44 >1000 >1000 >1000 1.3 .=1111111 na
na ......
Trr
FN Rbt22C4 94 >1000 >1000 >1000 2.2 === kW na
na
FN Rbt13D8 4.3 >1000 >1000 >1000 1.2 = =1000 na
na ......
......
*Rbt9E3.
*FN FN 2.6 na na na 0.6 na na na
:, .... . ....
*FN *Rbt10C3 3.4 na na na 0.7 na na na ......
Trr
*FN *Rbt10F7 7.0 na , na na 4.1 na na na :.:.:.
Igl Rbt11G11 31.3 >1000 39.8 >1000 4.1 3.4 na na
..
Igl Rbt12H4 18 92 16.7 >1000 8.5 11 , ,
...µ.
..=1000
' t
Igl Rbt18G7 2.3 21 0.5 >1000 I. I. 0.2
Igl Rbt13B4 2.9 8.8 0.3 >1000 .,-1000 MOO
.,'1000 '1000
- Rbt14C9 >1000 na na na ..j:#4*..........
a..........4g:..............i a...........Ak..............
..........::APP::........
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Example 4: Anti-MerTK inhibits human and mouse macrophage phagocytosis in
vitro
[0479] Efferocytosis assays were carried out to evaluate the in vitro
macrophage phagocytosis
inhibiting activity of anti-MerTK antibodies.
[0480] Briefly, efferocytosis, the phagocytosis of apoptotic cells, was
quantified using the
IncuCyte real time imaging platform. Apoptotic cells were labeled with pH-
sensitive probes
(pHrodo). pHrodo will only fluoresce in the acidic environment of the
phagolysosome once it has
been phagocytosed by a macrophage. Phagocytosis events were quantified as
total fluorescence
intensity (TFI) and normalized by the number of macrophages per well. The
maximum normalized
TFI observed was designated 100% Phagocytic Activity. The maximum phagocytosis
inhibition (0%
Phagocytic Activity) was designated as the autofluorescence generated by the
pHrodo-labeled
apoptotic cells alone in control wells without macrophages.
[0481] The efferocytosis assays demonstrated that humanized anti-MerTK
antibodies can inhibit
human macrophage phagocytosis of apoptotic cells (FIG. 4A, 4B & 4C). The
results suggested that
humanized antibody h13B4.v16 was the most potent inhibitor of phagocytosis
(TABLE 19). Further,
anti-MerTK antibody h13B4.v16 (13B4 Fully Humanized), an Ig-domain binding
antibody, was
found to be 5.2x more potent at inhibiting human macrophage phagocytosis
compared to anti-MerTK
antibody h10F7.v16 (10F7 Fully Humanized), a fibronectin-domain binding
antibody (TABLE 20;
FIG. 4D).
[0482] In addition, FIG. 4E shows the results of an efferocytosis assay
assessing the ability of
anti-MerTK antibodies to inhibit mouse macrophage phagocytosis. The results
demonstrated that the
anti-MerTK antibodies have the ability to block mouse macrophage phagocytosis
(FIG. 4E). Further,
the results showed that anti-MerTK antibody 14C9 mIgG2a LALAPG, an Ig-domain
binding
antibody, is 4.8x more potent at inhibiting mouse machrophage phagocytosis
compared to anti-
MerTK antibody hl0F7.v16 (10F7 Fully Humanized), a fibronectin-domain binding
antibody
(TABLE 21).
TABLE 19
Donor A Donor B Donor C Average (3 Donors)
Fully Max Max Max Max
Humanized IC50 Inhibition IC50 Inhibition IC50 Inhibition IC50 Inhibition
Antibodies (nM) (%) (111µ1) (%) (nM) (%) (111µ1) (%)
h13B4.v16 0.07 91 0.08 82 0.09 77 0.08 83
h10F7.v16 0.42 76 0.53 73 0.27 60 0.41 69
h10C3.v14 0.52 64 1.2 58 1.2 62 0.95 61
h9E3.FN.v16 0.59 58 NA NA 1.5 55 1.02 57
TABLE 20
Average (3 Donors)
Anti-MerTK Antibodies IC50 (nM) Max Inhibition (%)
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13B4 Fully Humanized 0.08 83
10F7 Fully Humanized 0.41 69
TABLE 21
Average
Anti-MerTK Antibodies IC50 (nM) Max Inhibition (%)
14C9 mIgG2a LALAPG 0.19 73
10F7 Fully Humanized 0.91 64
Example 5: Anti-MerTK inhibits the clearance of apoptotic cells in vivo
[0483] An apoptotic cell clearance assay was carried out to evaluate the in
vivo activity of anti-
MerTK antibodies (Seitz, H. M. et. al., Macrophages and dendritic cells use
different
Axl/Mertk/Tyro3 receptors in clearance of apoptotic cells, J Immunol. 178(9)
5635-5642 (2007)).
[0484] Briefly, 5-7 week-old C57BL/6 mice were injected intraperitoneally
with 0.2 mg/25 g
dexamethasone (Dex). Eight or twenty-four hours later, the thymus was isolated
and dissociated into a
single-cell suspension. Cells were stained with VAD-FMK-FITC (1:500 in PBS,
Promega, Cat#
G7461) to detect active caspase 3-positive apoptotic cells. Propidium iodide
was used to stain dead
cells (1:1000, Biochemika, Cat#: 70335). The cells were analyzed on a BD
FACSCalibur flow
cytometer. Accumulation of apoptotic cells were measured by VAD-FMK-FITC
single positive cells
(early apoptotic cells), and PINAD-FMK-FITC double positive cells (late
apoptotic cells). FIG. 5A
demonstrates that apoptotic cells accumulated 8 hours after Dex treatment and
were mostly cleared by
24 hours.
[0485] The clearance of apoptotic cells from the thymus is dependent on
MerTK expressed on
macrophages. Therefore, a panel of function blocking anti-MerTK antibodies was
tested for the ability
of each antibody to inhibit the clearance of apoptotic/dead cells. Anti-MerTK
(clone 14C9, mIgG2a,
LALAPG) but not the control antibody anti-gp120 (mIgG2a, LALAPG) blocked the
clearance of
apoptotic cells in the thymus 24 hours after Dex treatment (FIG. 5B).
Quantification of
apoptotic/dead cell accumulation in the thymus 24 hours after Dex injection in
mice treated with anti-
gp120 or anti-MerTK demonstrated that anti-MerTK antibodies blocked the
clearance of apoptotic
cells relative to the anti-gp120 control (FIG. 5C).
Example 6: Therapeutic effect of anti-MerTK antibodies in MC-38 syngeneic
tumor model
[0486] Tumor efficacy studies were carried out in the MC-38 syngeneic tumor
model to
determine whether anti-MerTK antibodies affect tumor growth.
[0487] Age-matched 6-8 week old female C57BL/6 mice were inoculated
subcutaneously in the
right unilateral flank with 1 x 105 MC-38 tumor cells suspended in Hanks's
Buffered Saline Solution
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(HBSS) and phenol red-free Matrigel (BD Bioscience). When tumors reached
volumes of 150-250
mm3 (day 0), mice were sorted into different treatment groups of n=10. Anti-
MerTK antibodies
(mIgG2a, LALAPG) or control anti-gp120 (mIgG2a, LALAPG) antibodies were
administered at
30 mg kg-' via intravenous (IV) injection on days 1 and 5, followed by intra-
peritoneal (IP) injection
on days 9 and 13. Anti-PDL1 antibody was administered at 30 mg kg' via IV
injection on day 1,
followed by IP injection on days 5, 9 and 13 at 5 mg kg'. Tumor volumes were
measured and
calculated twice per week using the modified ellipsoid formula 1/2 (length x
width2). Tumors
>2,000 mm3 were considered progressed.
[0488] In the tumor volume tracking plots, gray lines represent the tumor
size of animals that
were still in the study as of the date of data collection (FIGS. 6A & 6B). Red
lines represent animals
with ulcerated or progressed tumors that were euthanized and removed from
study (FIGS. 6A & 6B).
Red horizontal dashed lines indicate a doubling in tumor volume from the start
of treatment while
green horizontal dashed lines represent the smallest measureable tumor volume
(FIGS. 6A & 6B).
Animals with tumors in the area below the green dashed line were considered to
have had a complete
response.
[0489] As a monotherapy, anti-PDL1, an immune checkpoint inhibitor,
exhibited moderate anti-
tumor activity (FIGS. 6A-6D). Changes in individual tumor size (FIGS. 6A & 6B)
and mean tumor
size (FIGS. 6C & 6D), were measured over time for each treatment group.
Combination treatment
with anti-MerTK antibodies greatly enhanced the anti-tumor efficacy of the
anti-PDL1 antibodies
(FIGS. 6A-6D).
[0490] In the normal physiological context of solid tumors, the rapid
removal of dying tumor
cells by MerTK-expressing tumor associated macrophages (TAMs) is
immunologically silent.
Without being bound by theory, it is believed that blockade of MerTK,
accomplished in the above-
described experiments with anti-MerTK antibodies, could activate the innate
proinflammatory
response, which in turn could further enhance the adaptive T cell response
unleashed by anti-PD-1
therapy.
Example 7: Anti-MerTK antibody reduces clearance of apoptotic thymocytes in
vitro and in vivo
[0491] Efferocytosis assays were carried out to evaluate the in vitro
macrophage phagocytosis
inhibiting activity of an anti-MerTK antibody (clone 14C9, reformatted into a
mIgG2a, LALAPG
framework).
[0492] For in vitro efferocytosis assays, thymus tissue was harvested from
4-6 week old
C57BL/6N mice and minced to yield a single-cell suspension. Apoptosis of
thymocytes was induced
by 21.1M dexamethasone at 37 C for 5 hours. Membrane integrity and exposure
of phosphatidylserine
on cell surfaces were assessed using APC Annexin V Apoptosis Detection Kit
with PI (Biolegend).
Apoptotic thymocytes were labeled with 1 ug/m1 pHrodo Red succinimidyl ester.
Macrophages were
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pre-incubated with 30 lag/m1 control antibody or anti-MerTK 14C9 (mIgG2a
LALAPG) one hour
prior to adding pHrodo Red-labeled apoptotic cells. pHrodo will only fluoresce
in the
acidic environment of the phagolysosome once it has been phagocytosed by a
macrophage. After 45
minutes incubation, the remaining apoptotic cells were washed away, and
macrophages were labeled
with FITC-conjugated anti-CD lib antibody (eBioscience, clone M1/70). After
fluorescence images
were taken, the cells were detached from the cell culture plate for
quantification by FACS analysis.
[0493] For in vivo efferocytosis assays, 5-7 week-old C57BL/6N mice were
dosed with 20 mg/kg
anti-MerTK 14C9 (mIgG2a LALAPG) antibody and then injected intraperitoneally
with 0.2 mg/25 g
dexamethasone (Dex) one hour later. Eight or twenty-four hours later, the
thymus was isolated and
dissociated into a single-cell suspension. Cells were stained with VAD-FMK-
FITC (1:500 in PBS,
Promega, Cat# G7461) to detect active caspase 3-positive apoptotic cells.
Propidium iodide was used
to stain dead cells (1:1000, Biochemika, Cat#: 70335). The cells were analyzed
on a BD
FACSCalibur flow cytometer. Accumulation of apoptotic cells were measured by
VAD-FMK-FITC
single positive cells (early apoptotic cells) and PI/VAD-FMK-FITC double
positive cells (late
apoptotic cells).
[0494] In the in vitro efferocytosis assay, anti-MerTK 14C9 (mIgG2a LALAPG)
substantially
reduced the uptake of apoptotic thymocytes by peritoneal macrophages (FIG.
7B). Moreover, in the
in vivo assays, anti-MerTK 14C9 (mIgG2a LALAPG) effectively inhibited the
clearance of apoptotic
thymocytes in mice treated with dexamethasone (FIG. 7C & 8B). This in vivo
result was consistent
with the defective efferocytosis observed in MerTK deficient mice (Scott, R.S.
etal. Phagocytosis and
clearance of apoptotic cells is mediated by MER. Nature 411, 207-211(2001)),
demonstrating the
functional effectiveness of the anti-MerTK antibody.
Example 8: Anti-MerTK antibody inhibits ligand-mediated MerTK signaling
[0495] MerTK ligand-dependent AKT phosphorylation was measured to evaluate
the effect of
anti-MerTK antibody on ligand-mediated MerTK signaling.
[0496] Briefly, 1774A.1 mouse macrophages from an exponentially growing
culture were seeded
at a density of 2.0 x 105 cells/well on a 96-well plate in RPMI medium + 10%
FBS. The following
day, cells were washed with 200 ILtL of serum free RPMI twice and incubated in
200 ILtL of serum free
RPMI for 4 hours. After serum starvation, 10 1.1g/mL recombinant human GAS6-Fc
protein, which is a
ligand for MerTK, was added and incubated for 20 minutes. Phospho-AKT (pAKT)
measurements
were taken from treated cell lysates using the Phospho-AKT-1 (5er473) HTRF Kit
(Cisbio,
#63ADK078PEG) following the manufacturer's instructions (standard protocol for
two-plate assay
protocol in 20 ILtL final volume). The AKT phosphorylation assay demonstrated
that anti-MerTK
antibody potently inhibits ligand-mediated MerTK signaling compared to an
isotype control, as
measured by pAKT activity in macrophages (FIG. 8A).
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Example 9: Effect of anti-MerTK antibody on tumor-associated macrophages
[0497] MerTK expression and distribution studies were carried out in tumor
associated
macrophages (TAMs), one of the most abundant tumor infiltrating immune cells.
To isolate TAMs,
tumors were harvested and dissociated into single cell suspensions. Live cells
were enriched using
Lymphocyte M media (Cedarlane Labs). CD335+, Siglec F+, and anti-Ly6G/6C+
cells were labeled
with biotin-conjugated antibodies and depleted with anti-biotin MACSiBeadTM
Particles (Miltenyi
Biotec). TAMs were then purified with anti-F4/80 Microbeads (Miltenyi Biotec)
(FIG. 10A). The
purity of isolated TAMs was confirmed to be >90% as assessed by FACS (FIG.
10B). Fluorescence
microscopy was used to determine MerTK distribution in TAMs and the ability of
TAMs to clear
apoptotic cells (FIG. 8C & 8E). qPCR and transcriptome analyses were performed
to identify genes
that are differentially expressed in cells treated with anti-MerTK 14C9
(mIgG2a LALAPG) or a
control antibody (FIG. 9, 10, 11, & 13).
[0498] Analysis of MC38 syngeneic murine colon adenocarcinoma tumors
growing in wild-type
(W7) or Merte- mice showed specific expression of MerTK in TAMs (FIG. 8C). In
addition, TAMs
from MC38 tumors were able to engulf apoptotic cells and, importantly, anti-
MerTK 14C9 (mIgG2a
LALAPG) inhibited this uptake (FIG. 8E). These results demonstrate that MerTK
plays an important
role in the clearance of apoptotic cells by TAMs in the tumor microenvironment
and that treatment of
TAMs with anti-MerTK antibody inhibits this uptake.
[0499] Transcriptome analysis of TAMs from established MC38 tumors treated
with anti-MerTK
antibody was performed to determine the impact of MerTK inhibition on TAMs.
The transcriptome
analysis revealed that TAMs from mice treated with anti-MerTK 14C9 (mIgG2a
LALAPG) displayed
significant changes in gene expression as compared to TAMs treated with the
control antibody (FIG.
9A & 10C). Gene set enrichment analysis revealed Type I IFN response as the
most prominently up-
regulated gene signature (FIG. 9B & 10D). qPCR analysis confirmed the
upregulation of Ifnb 1 and
multiple interferon stimulated genes (ISGs) in TAMs from anti-MerTK 14C9
(mIgG2a LALAPG)
treated tumors (FIG. 9C & 11A). A significant increase of IFI\113 protein
(FIG. 9D) and concomitant
induction of ISGs was also observed in in tumor samples (FIG. 10E & 11B). The
upregulation of
Ifnb 1 expression was restricted to CD45+ immune cells and the basal level
expression of IF1\113 was
much higher in CD45+ immune cells than in CD45- cells. In addition, IF1\113
was significantly
upregulated in TAMs but not in DCs (FIG. 9E), and TAMs were considerably more
abundant than
DCs in the MC38 tumors (FIG. 14B).
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Example 10: Distribution of MerTK in human cancers
[0500] The distribution of MerTK expression in human cancers was determined
using expression
data from The Cancer Genome Atlas (TCGA). Expression data in TCGA samples were
obtained as
described by Daemen et al. (Daemen, A. et al. Pan-Cancer Metabolic Signature
Predicts Co-
Dependency on Glutaminase and De Novo Glutathione Synthesis Linked to a High-
Mesenchymal
Cell State. Cell Metab 28, 383-399 e389 (2018)). Gene expression in form of
RPKMs served as input
for TIMER software (Li, T. et al. TIMER: A Web Server for Comprehensive
Analysis of Tumor-
Infiltrating Immune Cells. Cancer Res 77, el08-e110 (2017)) to calculate
relative levels of six tumor-
infiltrating immune subsets. It was confirmed that MerTK was not part of the
signatures used to
estimate immune set abundance. Pearson correlation coefficients between gene
expression level and
immune cell type estimates were computed for each cell type and indication. In
human cancers,
MerTK expression exhibited greater correlation with the abundances of TAMs
compared to other
immune cell types (FIG. 8D), consistent with MerTK being expressed by TAMs.
Example 11: Anti-MerTK antibody induces the local Type I IFN response in the
tumor
microenvironment
[0501] The relationship between anti-MerTK antibody treatment and the Type
I IFN response
was investigated. Briefly, female C57BL/6 mice were inoculated subcutaneously
in the right
unilateral flank with 1 x 105 MC38 tumor cells suspended in Hanks's Buffered
Saline Solution
(HBSS) and phenol red-free Matrigel (1:1 v/v) (BD Bioscience) and then treated
with 20 mg/kg anti-
MerTK 14C9 (mIgG2a LALAPG) antibody or control antibody. Three days after
treatment, tumors
were homogenized in PBS supplemented with Halt Tm Protease and Phosphatase
Inhibitor Cocktail
(ThermoFisher Scientific) in gentleMACS M Tubes (Miltenyi Biotec) using
gentleMACS Dissociator
(Miltenyi Biotec) following the manufacturer's protocol. For every 100 mg of
tumor tissue, 500 iaL of
buffer was used. Tumor homogenates were clarified by centrifugation at 12,000
xg for 20 minutes at 4
C. Homogenates were normalized based on total protein concentrations
determined by BCA Protein
Assay Kit (Piece). IFN- 13 and CCL7 (MCP-3) were assayed using High
Sensitivity Mouse IFN Beta
ELISA Kit (PBL Assay Science) and Mouse MCP-3 Instant ELISA Kit (Invitrogen),
respectively.
Other cytokines/chemokines were assayed using MILLIPLEX MAP Mouse
Cytokine/chemokine
Magnetic Beads Penal-Premixed 15-Plex and 32-Plex (Millipore).
Cytokine/chemokine results were
expressed as pg/mg of total protein in tumor homogenate.
[0502] Type I IFNs activate autocrine and/or paracrine production of
cytokines and chemokines
that modulate innate and adaptive immune responses. In line with this, protein
levels of the cytokines
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or chemokines CCL3, CCL4, CCL5, CCL7, and CCL12 in anti-MerTK antibody treated
tumor
homogenates were observed (FIG. 13A) The type I IFN response appeared to be
restricted to the
tumor site, as no significant changes in ISG expression were found in
peripheral blood mononuclear
cells (PBMCs) collected from tumor bearing mice treated with anti-MerTK
antibody (FIG. 13B).
Significant changes in the expression of cytokines that were previously
reported to be linked to
MerTK activation, including IL10, TGF131, IL6 and IL12a (FIG. 13C) were not
observed. In
summary, these data demonstrate that anti-MerTK antibodies can induce the
local Type I IFN
response in the tumor microenvironment.
Example 12: Anti-MerTK antibody enhances antitumor immunity
[0503] Given that anti-MerTK antibody induced a type I IFN response and
that Type I IFNs
positively regulate various aspects of antigen-presenting cells (APCs), an
antigen presentation assay
was performed to determine whether antigen presentation by TAMs and tumor-
associated DCs is
enhanced by anti-MerTK antibody. Briefly, female C57BL/6 mice were inoculated
subcutaneously in
the right unilateral flank with 5 x 106 MC38.0VA tumor cells suspended in
Hanks's Buffered Saline
Solution (HBSS) and phenol red-free Matrigel (1:1 v/v) (BD Bioscience). When
tumors reached
volumes of 100-150 mm3 (day 0), mice were administered anti-MerTK 14C9 (mIgG2a
LALAPG)
antibody or control antibody anti-gp120 via intraperitoneal (IP) injection at
a dose of 20 mg/kg.
Tumors were later analyzed for antigen presentation enhancement. In the
MC38.0VA tumor model,
H-2K' bound OVA-derived SIINFEKL peptide can be readily detected for
monitoring antigen
presentation. The anti-H-2K"-SIINFEKL (Biolegend, clone 25-D1.16) was used to
specifically detect
OVA-derived peptide SIINFEKL bound to MHC class I H-2K', but not unbound H-2K'
or H-2K'
bound to other peptides.
[0504] Anti-MerTK antibody significantly increased the level of H-2K"-
SIINFEKL complex on
TAMs (FIG. 12A). CD86, a costimulatory molecule for T cell activation, was
also elevated in TAMs
but not DCs (FIG. 12A). A downregulation of CD206, an "M2-like" macrophage
marker, on TAMs
after anti-MerTK antibody treatment was also observed (FIG. 14C). These
findings suggest that anti-
MerTK antibody induces an immunogenic reprogramming of tumor microenvironment,
which in turn
could enhance the adaptive T cell response.
[0505] Tumor-infiltrating lymphocyte (TIL) clonality reflects the frequency
of T cells with a
specific TCR chain usage at the tumor site. To determine whether anti-MerTK
antibody treatment
affects clonal expansion of antigen-specific TILs, tumor-infiltrating T cells
were enriched using
Dynabeads Mouse Pan T Kit (ThermoFisher Scientific). Genomic DNA from enriched
T cells was
extracted using AllPrep DNA/RNA/Protein Mini Kit (Qiagen) and subjected to
TCRI3 CDR3
sequencing using the Immunoseq platform at survey level (Adaptive
Biotechnologies). Sequencing
results were analyzed using ImmunoSEQ Analyzer (Adaptive Biotechnologies).
Clonality scores were
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calculated as 1-(entropy)/10g2(number of productive unique sequences), where
the entropy takes into
account the varying clone frequency.
[0506] Anti-MerTK 14C9 (mIgG2a LALAPG) treatment led to a significant
increase in TIL
clonality (FIG. 12B), indicating clonal expansion of antigen-specific TILs. In
addition, anti-MerTK
14C9 (mIgG2a LALAPG) treatment increased the frequency of total CD8+ T cells,
as well as antigen
specific CD8+ T cells, e.g., T cells that recognize p15e, an endogenous
antigen presented by MC38
tumor cells (FIG. 12C). Therefore, MerTK blockade enhances the immune
recognition of tumor cells
and tumor-specific CD8+ T response.
Example 13: Anti-MerTK antibody is effective in combination with anti-PD-1,
anti-PD-L1, and
gemcitabine
[0507] To further characterize the effectiveness of anti-MerTK antibody as
a combination
therapy, tumor growth assays were performed as previously described. Briefly,
female C57BL/6 mice
were inoculated subcutaneously in the right unilateral flank with 1 x 105 MC38
tumor cells suspended
in Hanks's Buffered Saline Solution (HBSS) and phenol red-free Matrigel (1:1
v/v) (BD Bioscience).
On predetermined days post inoculation, mice were administered (1) the anti-
MerTK antibody as a
monotherapy (FIG. 15A); (2) anti-MerTK antibody and anti-PD-Li antibody as a
combination
therapy (FIG. 15B); or (3) anti-MerTK antibody, anti-PD-1 antibody, and the
chemotherapeutic
gemcitabine as a combination therapy (FIG. 15C). Anti-MerTK 14C9 (mIgG2a
LALAPG) was
administered at 20 mg/kg, anti-PD-Li was administered at 10 mg/kg, anti-PD1
was administered at 8
mg/kg, and gemcitabine was administered at 120 mg/kg. Treatments were
administered either at an
early stage of tumor progression (FIG. 15A) or when tumors were fully
established (FIG. 15B &
15C).
[0508] When treatment started at the early stage of tumor progression,
single-agent anti-MerTK
antibody was able to significantly reduce the tumor growth (FIG. 15A). In
comparison, in the
intervention setting of treating fully established tumors, anti-MerTK antibody
or anti-PD-Li antibody
alone had a marginal effect (FIG. 15B). In contrast, simultaneous treatment
with anti-MerTK
antibody and anti-PD-Li antibody exhibited a robust antitumor effect (FIG.
15B). Similarly,
treatment with anti-MerTK antibody significantly improved the efficacy of an
antibody targeting PD-
1 (the receptor for PD-L1) (FIG. 15C). The chemotherapy drug gemcitabine
moderately improved
anti-PD-1 antibody therapy. However, addition of anti-MerTK antibody to the
combination therapy of
gemcitabine plus anti-PD-1 antibody resulted in complete regression of all
treated tumors (FIG. 15C).
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Example 14: Anti-MerTK antibody antitumor effect depends on the presence of
functional STING
in the tumor host
[0509] To explore the role of Type I IFN signaling in anti-MerTK-induced
antitumor immune
responses, a functional neutralizing antibody against IFNAR1 (anti-IFNAR1
clone MAR1-5A3
BioXCell) was used to interfere with Type I IFN signaling and tumor growth
assays were performed
as previously described. Briefly, female C57BL/6 mice were inoculated
subcutaneously in the right
unilateral flank with 1 x 105 MC38 tumor cells suspended in Hanks's Buffered
Saline Solution
(HBSS) and phenol red-free Matrigel (1:1 v/v) (BD Bioscience). On
predetermined days post
inoculation, mice were administered (1) the anti-MerTK 14C9 (mIgG2a LALAPG)
antibody as a
monotherapy; (2) the anti-IFNAR1 antibody as a monotherapy; (3) anti-MerTK
14C9 (mIgG2a
LALAPG) and anti-PD-Li antibody as a combination therapy; or (4) anti-MerTK
14C9 (mIgG2a
LALAPG), anti-PD-Li antibody, and anti-INFAR1 antibody as a combination
therapy.
[0510] Anti-IFNAR1 antibody treatment completely abolished the modulation
of ISGs by
MerTK blockade (FIG. 16A). Blocking type I IFN signaling also negated the
antitumor activity of
anti-MerTK 14C9 (mIgG2a LALAPG) either as a single agent (FIG. 17A) or in
combination with
anti-PD-Li (FIG. 16B). These results demonstrate that the antitumor effect of
anti-MerTK antibody
depends on intact type I IFN signaling.
[0511] The STING pathway has emerged as a key signaling mechanism that
drives the antitumor
type I IFN response (Woo, S.R. et al. STING-dependent cytosolic DNA sensing
mediates innate
immune recognition of immunogenic tumors. Immunity 41, 830-842 (2014); Deng,
L. etal. STING-
Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-
Dependent
Antitumor Immunity in Immunogenic Tumors. Immunity 41, 843-852 (2014)). To
determine the role
of STING signaling for the antitumor effect of MerTK blockade, tumor studies
with WT and STING-
defective (Sting') mice were carried out. In contrast to the WTmice, no
upregulation of ISGs was
detected in Stinggt/gt mice after anti-MerTK antibody treatment (FIG. 17B).
Furthermore, the
antitumor effect of MerTK inhibition was lost in the absence of functional
STING in mice (FIG.
17C). These data demonstrate that the antitumor effect of anti-MerTK antibody
depends on the
presence of functional STING in the host.
Example 15: Anti-MerTK antibody antitumor effect depends on the presence of
functional cGAS in
tumor cells
[0512] Cytoplasmic DNA transfection experiments were carried out to
evaluate the effect of
STING and cGAS on the anti-MerTK antibody antitumor effect. Briefly, WT bone
marrow-derived
macrophages (BMDMs), Stinggt/gt BMDMs, WT J774A.1 macrophages, and cGAS-/-
J774A.1
macrophages were transfected with Herring testes-DNA (HT-DNA) using
lipofectamine 3000
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(Invitrogen) and then irradiated by 250 mJ/cm2UV-C using a UV crosslinker
(Stratagene) to induce
apoptosis and the resulting amount of IFN-beta protein was measured using the
High Sensitivity
Mouse IFN Beta ELISA Kit (PBL Assay Science). Functional cGAS and STING were
required in
macrophages for IFNfl induction in response to exogenously delivered cytosolic
DNA through
liposome-mediated transfection (FIG. 18A & 18B). Western blot analysis of cGAS
and STING
expression in MC38 tumor cells and J774A.1 macrophages determined that J774A.1
macrophages
express cGAS and STING, while MC38 tumor cells only express cGAS (FIG. 18C).
Consistent with
a lack of STING expression, MC38 cells themselves did not produce any
detectable IFNfl after UV
radiation (FIG. 18C & 19A). IFNfl was induced when UV-irradiated tumors cells
were co-cultured
with WT but not Stinggt/gt macrophages (FIG. 19A).
[0513] In another experiment, dying tumor cells were transfected with DNA
as described above,
cocultured with macrophages for 24 hours, and IFNfl protein levels in culture
supernatant were
determined using the High Sensitivity Mouse IFN Beta ELISA Kit (PBL Assay
Science).
Macrophages deficient in cGAS were still able to produce IFNfl when co-
cultured with dying tumor
cells (FIG. 19B). To investigate whether the tumor cells were providing
functional cGAS to the
macrophages, resulting in IFN-beta expression, cGAS-/- MC38 cells were tested
for the ability to
induce IFN-beta expression. This showed that cGAS-/- MC38 cells were unable to
stimulate IFNfl
production, regardless of the genotype of macrophages (FIG. 19A & 19B). These
results support a
model wherein STING in macrophages is trans-activated by tumor-derived cGAS.
[0514] To investigate the significance of tumor-derived cGAS in trans-
activating STING in vivo,
we carried out tumor studies with cGAS-/- MC38 or AB22 tumor cells. Briefly,
C57BL/6N mice were
inoculated with 1 x 105 WT or cGAS-/- MC38 cells or BALB/c mice were
inoculated with 1 x 10 WT
or cGAS-/- AB22 cells then treated with anti-MerTK 14C9 (mIgG2a LALAPG) or
control antibody as
described in Example 11. For early stage tumor investigation, mice were
administered anti-MerTK
14C9 (mIgG2a LALAPG) or control antibody 4 days after inoculation (FIG. 19C)
or anti-MerTK
14C9 (mIgG2a LALAPG), anti-PD-L1, or control antibody 4, 7, and 10 days after
inoculation with
tumor cells (FIGS. 19D & 19E). For established tumor investigation, mice were
administered anti-
MerTK 14C9 (mIgG2a LALAPG) in combination with anti-PD-Li or control antibody
18, 22, 26, and
30 days after inoculation (FIG. 18E), or tumors were grown to volumes of 100-
150 mm3, and then
mice were administered anti-MerTK 14C9 (mIgG2a LALAPG) or control antibody
that day (FIG.
18D).
[0515] After anti-MerTK antibody treatment the type I IFN response observed
in MC38 tumors
was completely lost in cGAS-/- MC38 tumors (FIG. 19C). Similar results were
obtained in
mesothelioma AB22 tumors (FIG. 18D). Importantly, cGAS deficiency rendered
tumors resistant to
single-agent treatment of anti-MerTK antibody or anti-PD-Li antibody in early
tumor progression
setting (FIGS. 19D & 19E) or to combination therapy when treating fully
established tumors (FIG.
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18E). Therefore, the anti-MerTK antibody antitumor effect depends on the
presence of functional
cGAS in tumor cells.
Example 16: Anti-MerTK antibody antitumor effect potentially depends on the
presence of gap
junctions between tumor cells and macrophages
[0516] It is known that the activation of cGAS leads to production of
cGAMP. To determine if
tumor cell-derived cGAMP is responsible for the activation of STING in immune
cells, protein
quantification by LC-MS/MS was used to measure cGAMP production in WT and cGAS-
/- MC38
tumor cells transfected with DNA. cGAMP increased following transfection with
HT-DNA in WT
tumor cells, but cGAS-/- tumor cells lost the capacity to generate cGAMP in
response to cytosolic
DNA (FIG. 18F). To determine if tight junctions facilitate tumor cell-derived
cGAMP transmission
into macrophages, dye transfer assays and IFN-beta transfer assays between
tumor and macrophage
cells were performed. For dye transfer assays, donor cells (WT MC38 tumor
cells, Cx43-/- MC38
tumor cells, or J774A.1 macrophages) were stained with 0.5 1.1g/m1Calcein-AM
dye (ThermoFisher)
in PBS at 37 C for 30 minutes and washed extensively with culture medium to
remove free dye.
Calcein-loaded donor cells were co-cultured with recipient cells (WT or Cx43-/-
MC38 tumor cells) at
a ratio of 3:1 for 4-5 hours. Cells were analyzed by FACS to assess dye
transfer. To increase Cx43
expression, J774A.1 macrophages were stimulated with 0.5 itg/ml LPS
(Invivogen) overnight before
their use for dye transfer experiment. PE-Texas Red conjugated anti-CD1lb
(ThermoFisher) was used
to distinguish macrophages from tumor cells.
[0517] Cx43 is the most ubiquitously expressed connexin family protein
(Cx), which assemble to
form gap junctions between neighboring cells. Loss of Cx43 abolished the dye
transfer between
MC38 cells (FIG. 20B & 20C), confirming Cx43 is the key connexin for forming
functional gap
junctions. The dye transfer experiment also showed Cx43-dependent
intercellular communication
between macrophages and MC38 tumor cells (FIG. 20D).
[0518] In another experiment, DNA was transfected into WT or Cx43-/- MC38
tumor cells to
induce the production of cGAMP. After DNA transfection, 5 x 105 tumor cells
were co-cultured with
x 105 LPS-treated J774A.1 macrophages for 24 hours to allow cGAMP transfer.
IFN13 protein in
culture supernatant was measured with the High Sensitivity Mouse IFN Beta
ELISA Kit (PBL Assay
Science). Since MC38 tumor cells are not able to produce IFN13 due to lack of
STING expression, the
production of IFN13 reflects a productive transfer of cGAMP from tumor cells
to macrophages. DNA-
transfected WT MC38 cells, but not Cx43-/- MC38 cells, induced IFN13
production (FIG. 21B). Given
that WT and Cx43-/- MC38 cells expressed similar level of cGAS (FIG. 20A), the
failed induction of
IFN13 by Cx43-/- MC38 cells is likely due to the defective gap junctions.
Collectively, these data
support the possibility of a gap junction-dependent transfer of cGAMP from
tumor cells to
macrophages.
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[0519] WT and Cx43-/- MC38 tumor cells were further investigated to
determine whether
defective gap junctions abolish the antitumor effect of anti-MerTK 14C9
(mIgG2a LALAPG) in this
model. Briefly, C57BL/6N mice were inoculated with Cx43-/- MC38 cells as
described in Example
11 and treated with anti-MerTK 14C9 (mIgG2a LALAPG) 4 days later. After anti-
MerTK antibody
treatment, unlike WT MC38 tumors, no significant changes in the expression of
ISGs in Cx43-/- MC38
tumors were observed (FIG. 21C).
[0520] The effect of Cx43 loss on the anti-MerTK antibody antitumor effect
was also
investigated. Briefly, C57BL/6N mice were inoculated with 1 x 105 WT or cGAS-/-
MC38 cells or
BALB/c mice were inoculated with 1 x 107 WT or Cx43-/- MC38 cells then treated
with anti-MerTK
14C9 (mIgG2a LALAPG) or control antibody as described in Example 11. Mice were
administered
anti-MerTK 14C9 (mIgG2a LALAPG) and anti-PD-Li as a combination therapy or
control antibody
at 14, 18, 22, and 26 days after inoculation with tumor cells. Cx43-/- MC38
tumors became resistant to
the combination therapy of anti-MerTK 14C9 (mIgG2a LALAPG) and anti-PD-Li
(FIG. 20E).
Collectively, these results demonstrate that anti-MerTK antibody is effective
at treating tumors and
that the effectiveness of anti-MerTK antibody is dependent on the presence of
host STING, tumor-
derived cGAS, and tight junctions between tumor and macrophage cells.
Example 17: Anti-MerTK antibody blocks ongoing clearance of apoptotic cells by
tumor associated
macrophages (TAMS)
[0521] Cell free DNA (cfDNA) in blood circulation is released by damaged or
dead cells (Wan,
J.C.M. etal. (2017) Nat. Rev. Cancer 17:223-238). In cancer patients or tumor
bearing mice, a
subpopulation of cfDNA is tumor-derived, called circulating tumor DNA (ctDNA).
In this Example,
a SNP was utilized to distinguish host-derived cfDNA from tumor-derived ctDNA
in an MC38 tumor
model to investigate the effect of anti-MerTK antibody treatment.
[0522] MC38 tumor cells were inoculated into C57BL/6J mice and tumors were
allowed to
establish. Anti-MerTK or control antibody was administered after tumors were
established. Three
days post treatment, whole blood was collected by cardiac puncture into Cell-
free DNA BCT tubes
(Streck). Plasma was obtained by a double spin procedure (1,600 g for 10
minutes, separation,
followed by 16,000 g for 10 minutes). cfDNA (12.5 L for 200 L of plasma) was
obtained using
MagMAXTM Cell-Free DNA Isolation Kit (ThermoFisher Scientific) following the
manufacturer's
protocol.
[0523] To assay the levels of host-derived cfDNA and MC38-derived ctDNA,
multiplexing
droplet digital PCR (Bio-Rad Laboratories) was performed using an assay
containing primers and
probes targeting SNPs of gene Jmjdlc (r513480628, ThermoFisher Scientific).
C57BL/6J mice and
MC38 cells express a "T" and a "C" allele at this locus, respectively. For
droplet digital PCR, 4 L of
isolated cfDNA was used in each 20 L-reaction, and each sample was analyzed
in duplicates.
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Sample analysis was performed using QuantaSoft software (Bio-Rad
Laboratories), and target DNA
(copies/gL of plasma) was calculated as the quantitative outcome. Size of
isolated cfDNA was also
confirmed to be predominantly ¨170bp using Agilent Bioanalyzer 2100.
[0524] MC38 tumor cells were inoculated into C57BL/6J mice as described
above, and anti-
MerTK or control antibody was administered after tumors were established.
Three days after anti-
MerTK treatment, a significant increase of ctDNA in the plasma of tumor-
bearing mice was detected
(FIG. 22A). Anti-MerTK also increased the level of host-derived cfDNA in blood
circulation (FIG.
22B). These results clearly demonstrate that in tumor microenvironment anti-
MerTK was able to
block the ongoing clearance of apoptotic cells by TAMs.
Example 18: Analysis of anti-MerTK antibody binding affinity and epitope
mapping
[0525] For binding affinity determinations of anti-MerTK antibodies of the
present disclosure as
a control along with commercial MerTK antibodies, Surface Plasmon Resonance
(SPR) measurement
with a BIAcoreTm-T200 instrument was used. First, two rabbit antibodies (Y323
and 10g86D21F11)
and the anti-MerTK antibody h13B4.v16 were captured by protein A sensor chip,
and eight mouse
antibodies (A3KCAT, 2D2, 7E5G1, 7N-20, 590H11G1E3, MAB891, MAB8911 and MAB8912-
100)
were captured by goat anti-mouse IgGs sensor chip respectively on each flow
cell to achieve
approximately 100RU. Three-fold serial dilutions of human MerTK (0.4nM to
100nM) were injected
in HBS-EP buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA and 0.05% v/v
surfactant P20)
at 25 C with a flow rate of 50 1/min to record the binding response as a
function of time. The
sensorgram was fitted with one-to-one Langmuir binding model to calculate
association rates (kon)
and dissociation rates (koff) (BIAcore T200 evaluation software version 2.0).
The binding affinity
(equilibrium dissociation constant (KD)) was calculated as the ratio koff/kon.
[0526] As shown in FIG. 23, only 4 out of 10 selected commercial antibodies
showed binding to
human MerTK. The results indicated a binding affinity to human MerTK of 0.4nM
for Y323, 6.8nM
for A3KCAT, 7.6nM for 590H11G1E3, 17.3nM for MAB8912-100 and 1.6nM for
h13B4.v16, while
the remaining antibodies showed no binding. FIG. 23 shows that Y323 is a
higher affinity antibody
than h13B4.v16, including having about a 12-fold higher on-rate (ka) and 3-
fold higher off-rate (kd)
compared to h13B4.v16. In addition, as noted above, FIGS. 3, 4A-4C, & Table 19
demonstrate that
h13B4.v16 possesses biological properties desired for an anti-MerTK antibody,
such as more potent
inhibition of efferocytosis. Accordingly, h13B4.v16 possesses unique binding
characteristics
including on and off rates, affinity, binding epitope, and the resulting
desired biological effects, e.g.,
efferocytosis, which make this antibody a particularly useful therapeutic
candidate.
[0527] These 4 antibodies (Y323, A3KCAT, 590H11G1E3 and MAB8912-100) were
further
assessed to determine whether their binding epitope competes with h13B4.v16
for binding human
MerTK. To conduct this experiment, the same BIAcoreTm-T200 instrument was
used, and the classic
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sandwich format was applied (FIG 24A). h13B4.v16 at 2ug/mL was first captured
by goat anti-
human Fab sensor chip, and then 50nM of human MerTK was injected at a flow
rate of 500/min in
HBS-EP buffer to record l' binding, followed by the rd binding with or without
the tested antibody
at lOug/mL of injection. If the rd binding was observed, the tested antibody
did not compete with the
lead molecule, and vice versa, if the rd binding was not observed, the tested
antibody did compete
with h13B4.v16.
[0528] The results indicated that only antibody Y323 competed with
h13B4.v16 for binding to
human MerTK (FIG. 24B). The remaining three antibodies did not compete with
h13B4.v16 for
binding to human MerTK (FIG. 24C).
[0529] Although the present disclosure has been described in some detail by
way of illustration
and example for purposes of clarity of understanding, the descriptions and
examples should not be
construed as limiting the scope of the present disclosure. The disclosures of
all patent and scientific
literature cited herein are expressly incorporated in their entirety by
reference.
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